U.S. patent application number 11/561011 was filed with the patent office on 2008-11-27 for succinic acid-producing bacterium and process for producing succinic acid.
This patent application is currently assigned to AJINOMOTO CO., INC.. Invention is credited to Keita FUKUI, Hiroyuki KOJIMA, Jun NAKAMURA.
Application Number | 20080293112 11/561011 |
Document ID | / |
Family ID | 35428402 |
Filed Date | 2008-11-27 |
United States Patent
Application |
20080293112 |
Kind Code |
A1 |
FUKUI; Keita ; et
al. |
November 27, 2008 |
SUCCINIC ACID-PRODUCING BACTERIUM AND PROCESS FOR PRODUCING
SUCCINIC ACID
Abstract
Coryneform bacterium is modified so that an activity of
acetyl-CoA hydrolase is decreased, and succinic acid is produced by
using the bacterium.
Inventors: |
FUKUI; Keita; (Kawasaki,
JP) ; NAKAMURA; Jun; (Kawasaki, JP) ; KOJIMA;
Hiroyuki; (Kawasaki, JP) |
Correspondence
Address: |
GREENBLUM & BERNSTEIN, P.L.C.
1950 ROLAND CLARKE PLACE
RESTON
VA
20191
US
|
Assignee: |
AJINOMOTO CO., INC.
Tokyo
JP
MITSUBISHI CHEMICAL CORPORATION
Tokyo
JP
|
Family ID: |
35428402 |
Appl. No.: |
11/561011 |
Filed: |
November 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/JP05/09232 |
May 20, 2005 |
|
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11561011 |
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Current U.S.
Class: |
435/145 ;
435/252.32 |
Current CPC
Class: |
C12N 9/16 20130101; C12P
7/46 20130101; C12Y 301/02001 20130101; C12N 15/77 20130101; C08G
63/16 20130101 |
Class at
Publication: |
435/145 ;
435/252.32 |
International
Class: |
C12P 7/46 20060101
C12P007/46; C12N 1/21 20060101 C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2004 |
JP |
2004-150658 |
Claims
1. A coryneform bacterium having a succinic acid-producing ability,
wherein said bacterium has been modified so that an activity of
acetyl-CoA hydrolase is decreased.
2. The coryneform bacterium according to claim 1, wherein the
acetyl-CoA hydrolase is a protein as described in the following (A)
or (B); (A) a protein having an amino acid sequence of SEQ ID NO:
45; or (B) a protein having an amino acid sequence of SEQ ID NO: 45
including substitution, deletion, insertion, or addition of one or
several amino acids, and having an acetyl-CoA hydrolase
activity.
3. The coryneform bacterium according to claim 1, wherein the
acetyl-CoA hydrolase activity has been decreased by disruption of
an acetyl-CoA hydrolase gene on a chromosome.
4. The coryneform bacterium according to claim 3, wherein the
acetyl-CoA hydrolase gene is a DNA as described in the following
(a) or (b): (a) a DNA comprising a nucleotide sequence of
nucleotide numbers 1037-2542 in SEQ ID NO: 44; or (b) a DNA that
hybridizes with a nucleotide sequence of nucleotide numbers
1037-2542 in SEQ ID NO: 44 or a probe that can be prepared for the
nucleotide sequence under stringent conditions, and encodes a
protein having an acetyl-CoA hydrolase activity.
5. The coryneform bacterium according to claim 1, which has been
further modified so that activities of one or both of
phosphotransacetylase and acetate kinase are decreased.
6. The coryneform bacterium according to claim 1, wherein said
bacterium has been further modified so that an activity of lactate
dehydrogenase is decreased.
7. The coryneform bacterium according to claim 1, wherein said
bacterium has been further modified so that an activity of pyruvate
carboxylase is increased.
8. A method for producing succinic acid, comprising allowing the
coryneform bacterium according to claim 1 or a treated product
thereof to act on an organic raw material in a reaction liquid
containing a carbonate ion, a bicarbonate ion, or carbon dioxide to
generate and accumulate succinic acid in the reaction liquid, and
collecting succinic acid from the reaction liquid.
9. The production method according to claim 8, wherein the
coryneform bacterium or treated product thereof is allowed to act
on the organic raw material under anaerobic conditions.
10. A method for producing a succinic acid-containing polymer,
comprising the steps of producing succinic acid by the method
according to claim 8 and polymerizing the obtained succinic acid.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a Continuation of International
Application No. PCT/JP2005/009232, filed May 20, 2005.
TECHNICAL FIELD
[0002] The present invention relates to a fermentation industry,
and to a process for efficiently producing succinic acid by a
fermentation method using a coryneform bacterium.
BACKGROUND ART
[0003] For production of non-amino organic acids including succinic
acid by fermentation, usually, anaerobic bacteria such as those
belonging to the genus Anaerobiospirillum or Actinobacillus are
used (Patent Document 1 or 2, and Non-Patent Document 1). The use
of anaerobic bacteria makes the yield of products high, while such
bacteria require many nutrients for proliferation and therefore,
there is a need for adding a large amount of organic nitrogen
sources such as corn steep liquor (CSL) to a medium. The addition
of abundant amounts of organic nitrogen sources not only leads to
an increase in cost of the medium but also leads to an increase in
cost of purification for isolating the product, which is
uneconomical.
[0004] Furthermore, a method, which comprises culturing aerobic
bacteria such as coryneform bacteria under an aerobic condition to
proliferate bacterial cells and then collecting and washing the
cells to use them as resting bacteria to produce succinic acid
without oxygen aeration, has been known (Patent Document 3 and 4).
This method is economical because the bacterial cells can grow
sufficiently in a simple medium, into which a small amount of
organic nitrogen is added for proliferation of bacterial cells, but
this method is still to be improved in terms of the amount of
generated succinic acid, the concentration thereof, and the
production rate thereof per bacterial cells as well as
simplification of production process, and the like.
[0005] Furthermore, when aerobic bacteria such as coryneform
bacteria are cultured under oxygen-limited conditions, organic
acids other than a desired substance such as lactic acid and acetic
acid are excessively accumulated as by-products, resulting in
suppressed growth of bacterial cells and significantly decreased
productivity in fermentation. In addition, excessive amounts of
counterions to neutralize the organic acids generated as
by-products are required, thereby resulting in being uneconomical.
To solve such problems, reduction in lactate generated as a
by-product has been performed by using a coryneform bacterium
having a reduced lactate dehydrogenase activity (Patent Document
5).
[0006] Even if the above-mentioned coryneform bacterium having a
reduced lactate dehydrogenase activity is used, a large amount of
acetic acid is generated as a by-product. As means for achieving
the reduction of acetic acid in a culture medium, there have been
known a method of enhancing expression of an acetic acid
assimilating gene (aceP) in a bacterium belonging to the genus
Escherichia (Patent Document 6), a method of enhancing expression
of a gene encoding ACE protein in a bacterium belonging to the
genus Escherichia (Patent Document 7), and the like. Those methods
are intended to reduce generation of acetic acid as a by-product by
actively assimilating acetic acid released into a culture medium.
Meanwhile, as methods of suppressing generation of acetic acid as a
by-product by suppressing the biosynthesis of acetic acid, there
have been known a method of producing succinic acid using
Escherichia coli in which phosphoacetyltransferase (pta) and
lactate dehydrogenase (ldh) are deficient (Patent Document 8), a
method of producing an amino acid using an enterobacterium in which
pyruvate oxidase (poxB) is deficient, and a method of producing
D-pantothenic acid using an enterobacterium in which pyruvate
oxidase (poxB) is deficient (Patent Document 9).
[0007] As enzymes responsible for assimilation of acetic acid in
coryneform bacteria, there have been reported acetate kinase (ack)
and phosphotransacetylase (pta) (Non-Patent Document 2). On the
other hand, it is assumed that not only the above-mentioned enzymes
but also a plurality of enzymes such as pyruvate oxidase (poxB)
(Patent Document 10), acylphosphatase (acp), aldehyde
dehydrogenase, and acetyl-CoA hydrolase are responsible for
generation of acetic acid, but a specific enzyme that contributes
to the synthesis of acetic acid has not been clarified. Therefore,
there has not been known a method of producing succinic acid using
a strain of a coryneform bacterium having a decreased acetic acid
biosynthetic enzyme.
[0008] Acetyl-CoA hydrolase is an enzyme to generate acetic acid
from acetyl-CoA and water (3.1.2.1) and the gene sequence in
Corynebacterium glutamicum has been predicted (Patent Document 11).
However, no report has been provided for cloning of the gene and
expression analysis of the gene, and its actual function has not
been clarified.
[0009] Patent Document 1: U.S. Pat. No. 5,143,833
[0010] Patent Document 2: U.S. Pat. No. 5,504,004
[0011] Patent Document 3: JP11-113588A
[0012] Patent Document 4: JP11-196888A
[0013] Patent Document 5: JP11-206385A
[0014] Patent Document 6: JP06-14781A
[0015] Patent Document 7: JP07-67683A
[0016] Patent Document 8: WO 99/06532
[0017] Patent Document 9: WO 02/36797
[0018] Patent Document 10: EP1096013A
[0019] Patent Document 11: EP1108790A
[0020] Non-Patent Document 1: International Journal of Systematic
Bacteriology, vol. 49, p 207-216, 1999
[0021] Non-Patent Document 2: Microbiology. 1999, February; 145 (Pt
2): 503-13
DISCLOSURE OF THE INVENTION
[0022] An object of the present invention is to provide a
coryneform bacterium having an improved succinic acid-producing
ability.
[0023] The inventors of the present invention have intensively
studied for solving the aforementioned problems, and as a result
found that succinic acid-producing ability is improved by
decreasing an activity of acetyl-CoA hydrolase in a coryneform
bacterium. Furthermore, they found that generation of acetic acid
as a by-product is reduced by decreasing activities of
phosphotransacetylase and acetate kinase in addition to the
acetyl-CoA hydrolase activity, thereby accomplished the present
invention.
[0024] That is, the present invention is as follows. [0025] (1) A
coryneform bacterium having a succinic acid-producing ability,
wherein said bacterium has been modified so that an activity of
acetyl-CoA hydrolase is decreased. [0026] (2) The coryneform
bacterium according to (1), wherein the acetyl-CoA hydrolase is a
protein as described in the following (A) or (B):
[0027] (A) a protein having an amino acid sequence of SEQ ID NO:
45; or
[0028] (B) a protein having an amino acid sequence of SEQ ID NO: 45
including substitution, deletion, insertion, or addition of one or
several amino acids, and having an acetyl-CoA hydrolase activity.
[0029] (3) The coryneform bacterium according to (1) or (2),
wherein the acetyl-CoA hydrolase activity has been decreased by
disruption of an acetyl-CoA hydrolase gene on a chromosome. [0030]
(4) The coryneform bacterium according to (3), wherein the
acetyl-CoA hydrolase gene is a DNA as described in the following
(a) or (b):
[0031] (a) a DNA comprising a nucleotide sequence of nucleotide
numbers 1037-2542 in SEQ ID NO: 44; or
[0032] (b) a DNA that hybridizes with a nucleotide sequence of
nucleotide numbers 1037-2542 in SEQ ID NO: 44 or a probe that can
be prepared from the nucleotide sequence under stringent
conditions, and encodes a protein having an acetyl-CoA hydrolase
activity. [0033] (5) The coryneform bacterium according to any one
of (1) to (4), which has been further modified so that activities
of one or both of phosphotransacetylase and acetate kinase are
decreased [0034] (6) The coryneform bacterium according to any one
of (1) to (5), wherein said bacterium has been further modified so
that an activity of lactate dehydrogenase is decreased. [0035] (7)
The coryneform bacterium according to any one of (1) to (6),
wherein said bacterium has been further modified so that an
activity of pyruvate carboxylase is increased. [0036] (8) A method
for producing succinic acid, comprising allowing the coryneform
bacterium according to any one of (1) to (7) or a treated product
thereof to act on an organic raw material in a reaction liquid
containing carbonate ion, bicarbonate ion, or carbon dioxide to
generate and accumulate succinic acid in the reaction liquid, and
collecting succinic acid from the reaction liquid. [0037] (9) The
production method according to (8), wherein the coryneform
bacterium or treated product thereof is allowed to act on the
organic raw material under anaerobic conditions. [0038] (10) A
method for producing a succinic acid-containing polymer, comprising
the steps of producing succinic acid by the method according to (8)
or (9) and polymerizing the obtained succinic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 shows the procedures for constructing plasmid
pBS3.
[0040] FIG. 2 shows the procedures for constructing plasmid
pBS4S.
[0041] FIG. 3 shows the procedures for constructing plasmid
pBS5T.
[0042] FIG. 4 shows the procedures for constructing plasmid
p.DELTA.ldh56-1.
[0043] FIG. 5 shows the procedures for constructing plasmid
pBS5T::.DELTA.ack.
[0044] FIG. 6 shows the procedures for constructing plasmid
pBS5T::.DELTA.pta-ack.
[0045] FIG. 7 shows the procedures for constructing plasmid
pBS5T::.DELTA.poxB.
[0046] FIG. 8 shows the procedures for constructing plasmid
pBS4S::.DELTA.ach.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0047] Hereinafter, embodiments of the present invention will be
described in detail.
<1> Coryneform Bacterium To Be Used In the Present
Invention
[0048] In the present invention, the term "coryneform bacterium"
includes a bacterium which had been classified as the genus
Brevibacterium but now classified as the genus Corynebacterium
(Int. J. Syst. Bacteriol., 41, 255 (1981)), and it also includes a
bacterium belonging to the genus Brevibacterium, which is very
closely related to Corynebacterium. Examples of such coryneform
bacteria include the followings.
[0049] Corynebacterium acetoacidophlilum
[0050] Corynebacterium acetoglutamicum
[0051] Corynebacterium alkanolyticum
[0052] Corynebacterium callunae
[0053] Corynebacterium glutamicum
[0054] Corynebacterium lilium
[0055] Corynebacterium melassecola
[0056] Corynebacterium thermoaminogenes
[0057] Corynebacterium herculis
[0058] Brevibacterium divaricatum
[0059] Brevibacterium flavum
[0060] Brevibacterium immariophilum
[0061] Brevibacterium lactofermentum
[0062] Brevibacterium roseum
[0063] Brevibacterium saccharolyticum
[0064] Brevibacterium thiogenitalis
[0065] Corynebacterium ammoniagenes
[0066] Brevibacterium album
[0067] Brevibacterium selinum
[0068] Microbacterium ammoniaphilum
[0069] In the present invention, the term "succinic acid-producing
ability" means an ability to accumulate succinic acid in a medium
when the coryneform bacterium of the present invention is cultured.
The succinic acid-producing ability may be a feature inherent to a
wild-type coryneform bacterium or a feature provided by
breeding.
[0070] To provide the succinic acid-producing ability by breeding,
there may be applied methods that have been employed in breeding of
coryneform bacteria, which include acquisition of metabolic
regulation mutant strains, creation of a recombinant strain having
an enhanced biosynthetic enzyme for a desired substance, and the
like (Amino Acid Fermentation, Japan Scientific Societies Press,
the first edition published on May 30, 1986, p 77-100). In these
methods, one or two or three or more features such as metabolic
regulation mutations and enhancement of biosynthetic enzymes for a
desired substance may be provided. Imparting properties such as
metabolic regulation mutations and enhancement of biosynthetic
enzymes may be combined.
[0071] Particularly preferable specific examples of a coryneform
bacterium having a succinic acid-producing ability include
Brevibacterium flavum MJ233.DELTA.ldh strain having decreased
lactate dehydrogenase activity (JP11-206385A), Brevibacterium
flavum MJ233/pPCPYC strain having enhanced activity of pyruvate
carboxylase or phosphoenol pyruvate carboxylase (WO 01/27258 and
JP11-196887A), Brevibacterium flavum MJ-233 (FERM BP-1497,
Brevibacterium flavum MJ-233 AB-41 (FERM BP-1498), Brevibacterium
ammoniagenes ATCC6872, Corynebacterium glutamicum ATCC31831, and
Brevibacterium lactofermentum ATCC13869. Since Brevibacterium
flavum may be currently classified as Corynebacterium glutamicum
(Lielbl, W., Ehrmann, M., Ludwig, W. and Schleifer, K. H.,
International Journal of Systematic Bacteriology, 1991, vol. 41, p
255-260), the aforementioned Brevibacterium flavum MJ-233 strain
and its mutant MJ-233 AB-41 strain, are defined as the same strains
as Corynebacterium glutamicum MJ-233 strain and Corynebacterium
glutamicum MJ-233 AB-41 strain, respectively.
<2> Construction of the Coryneform Bacterium of the Present
Invention
[0072] The coryneform bacterium of the present invention has the
above-mentioned succinic acid-producing ability and has been
modified so that an activity of acetyl-CoA hydrolase is
decreased.
[0073] In breeding of the coryneform bacterium of the present
invention, the provision of succinic acid-producing ability and the
modification to decrease the acetyl-CoA hydrolase (EC 3.1.2.1)
activity may be performed in any order.
[0074] The term "acetyl-CoA hydrolase (ACH) activity" means an
activity to catalyze the reaction to generate acetic acid from
acetyl-CoA and water. The term "modified so that activity of
acetyl-CoA hydrolase is decreased" means that an activity of
acetyl-CoA hydrolase is decreased as compared to a specific
activity of an unmodified strain such as a wild-type coryneform
bacterium. The ACH activity is preferably decreased to 50% or less
per bacterial cells, more preferably 30% or less, further more
preferably 10% or less per bacterial cells as compared to an
unmodified strain. Here, examples of a wild-type coryneform
bacterium to be used as a control include Brevibacterium
lactofermentum ATCC13869 (wild-type strain) and Brevibacterium
lactofermentum .DELTA.ldh strain (unmodified strain). The activity
of acetyl-CoA hydrolase can be determined according to the method
of Gergely, J., et al. (Gergely, J., Hele, P. & Ramkrishnan, C.
V. (1952) J. Biol. Chem. 198 p 323-334). The term "decreased"
includes complete loss of the activity. The coryneform bacterium of
the present invention preferably has an acetyl-CoA hydrolase
activity lower than a wild-type or unmodified strain and more
preferably has improved accumulation of succinic acid as compared
to those strains.
[0075] Examples of acetyl-CoA hydrolase having the above-mentioned
activity include a protein having an amino acid sequence of SEQ ID
NO: 45. In addition, it may be a protein having an amino acid
sequence of SEQ ID NO: 45 including a substitution, deletion,
insertion or addition of one or several amino acids as long as it
has an acetyl-CoA hydrolase activity. Here, for example, the term
"several" means 2 to 20, preferably 2 to 10, more preferably 2 to
5.
[0076] The term "modified so that the acetyl-CoA hydrolase activity
is decreased" includes decrease in the number of molecules of
acetyl-CoA hydrolase per cell, decrease in the acetyl-CoA hydrolase
activity per molecule, and the like. Specifically, it is achieved
by making a gene encoding acetyl-CoA hydrolase on a chromosome
deficient, modification of an expression regulatory sequence such
as promoter or Shine-Dalgarno (SD) sequence, or the like. Examples
of acetyl-CoA hydrolase gene on a chromosome include a DNA having a
nucleotide sequence of nucleotide numbers 1037-2542 of SEQ ID NO,
44. It may be a DNA that hybridizes with the nucleotide sequence of
nucleotide numbers 1037-2542 of SEQ ID NO: 44 or a probe that can
be prepared from the nucleotide sequence under stringent conditions
as long as it encodes a protein having acetyl-CoA hydrolase
activity. The term "stringent conditions" means conditions in which
so-called specific hybrid is formed and non-specific hybrid is not
formed. It is difficult to clearly define the conditions by numeric
value, but examples thereof include conditions comprising washing
once, preferably twice or three times at salt concentration
corresponding to 1.times.SSC, 0.1% SDS, preferably 0.1.times.SSC,
0.1% SDS at 60.degree. C.
[0077] The acetyl-CoA hydrolase gene (hereinafter, referred to as
ach gene) can be cloned by synthesizing synthetic oligonucleotides
based on a sequence of Corynebacterium glutamicum registered in
GenBank (NCg12480 of GenBank Accession No. NC.sub.--003450
(complementary strand of 2729376 . . . 2730884 of
NC.sub.--003450)), and performing PCR using a chromosome of
Corynebacterium glutamicum as a template. Furthermore, there may
also be used a sequence of a coryneform bacterium such as
Brevibacterium lactofermentum having a nucleotide sequence
determined by recent genome project. Chromosomal DNA can be
prepared from a bacterium as a DNA donor by, for example, a method
of Saito and Miura (U. Saito and K. Miura, Biochem. Biophys. Acta,
72, 619 (1963), Experimental Manual for Biotechnology, edited by
The Society for Biotechnology, Japan, p 97-98, Baifukan Co., Ltd.,
1992) or the like.
[0078] The ach gene thus prepared or a part thereof can be used for
gene disruption. A gene to be used for gene disruption only needs
to have homology that is enough to cause homologous recombination
with an ach gene to be disrupted on a chromosomal DNA of a
coryneform bacterium (e.g. a gene having the nucleotide sequence of
nucleotide numbers 1037-2542 in SEQ ID NO: 44), so such a
homologous gene may also be used. Here, the homology that is enough
to cause homologous recombination is preferably not less than 70%,
more preferably not less than 80%, further more preferably not less
than 90%, particularly preferably not less than 95%. Further, a DNA
capable of hybridizing with the above-mentioned gene under
stringent conditions can cause homologous recombination. The term
"stringent conditions" refers to conditions under which a so-called
specific hybrid is formed and non-specific hybrid is not formed. It
is difficult to clearly define the conditions by numeric value, but
the conditions include, for example, conditions that comprise
washing once, preferably twice or three times at salt
concentrations corresponding to 1.times.SSC, 0.1% SDS, preferably
0.1.times.SSC, 0.1% SDS at 60.degree. C.
[0079] For example, by using the above-mentioned gene, a
deleted-form of ach gene, which is modified so as not to produce
acetyl-CoA hydrolase that normally functions by deleting a partial
sequence of the ach gene, is prepared, and a coryneform bacterium
is transformed with a DNA containing the gene to cause
recombination between the deleted-form of the gene and the gene on
a chromosome, to thereby disrupt the ach gene on a chromosome. Such
a gene disruption by gene substitution using homologous
recombination has already been established, and examples thereof
include a method using a linear DNA and a method using a plasmid
containing a temperature-sensitive replication origin (U.S. Pat.
No. 6,303,383 or JP05-007491A). Further, the above-mentioned gene
disruption based on gene substitution using homologous
recombination may also be performed using a plasmid having no
replication ability in a host.
[0080] For example, an ach gene on a chromosome of a host can be
substituted by a deleted-form of ach gene in accordance with the
following procedures. First, a plasmid for recombination is
prepared by inserting a temperature-sensitive replication origin,
deleted-form of ach gene, sacB gene encoding levansucrase and a
marker gene exhibiting resistance to such a drug as
chloramphenicol.
[0081] Here, sacB gene encoding levansucrase is a gene which is
used for efficiently selecting a strain in which a vector portion
has been excised from a chromosome (Schafer, A. et al., Gene 145
(1994) 69-73). That is, when levansucrase is expressed in a
coryneform bacterium, levan generated by assimilation of sucrose
acts lethally on the bacterium, so the bacterium cannot grow.
Therefore, if a bacterial strain in which a vector carrying
levansucrase remains on a chromosome is cultured on a
sucrose-containing plate, it cannot grow. As a result only a
bacterial strain from which the vector has been excised can be
selected on the sucrose-containing plate.
[0082] Genes each having the following sequences can be used as a
sacB gene or homologous gene thereof.
[0083] Bacillus subtilis: sacB GenBank Accession Number X02730 (SEQ
ID NO: 35)
[0084] Bacillus amyloliquefaciens: sacB GenBank Accession Number
X52988
[0085] Zymomonas mobilis: sacB GenBank Accession Number L33402
[0086] Bacillus stearothermophilus: surB GenBank Accession Number
U34874
[0087] Lactobacillus sanfranciscensis: frfA GenBank Accession
Number AJ508391
[0088] Acetobacter xylinus: lsxA GenBank Accession Number
AB034152
[0089] Gluconacetobacter diazotrophicus: lsdA GenBank Accession
Number L41732
[0090] A coryneform bacterium is transformed with the
above-mentioned recombinant plasmid. The transformation can be
performed in accordance with a transformation method which has been
previously reported. Examples of the method include, a method of
increasing permeability of a DNA by treating cells of a recipient
bacterium with calcium chloride as reported for Escherichia coli
K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and,
a method of preparing competent cells using proliferating cells for
introduction of DNA as reported for Bacillus subtilis (Duncan. C.
H., Wilson, G. A and Young, F. E, Gene, 1, 153 (1977)).
Alternatively, as reported for Bacillus subtilis, actinomycetes and
yeasts, a method of introducing a recombinant DNA into cells of a
DNA recipient bacterium (Chang. S. and Choen, S. N., Molec. Gen.
Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M., and Hopwood, O.
A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G.
R., Proc. Natl. Acad. Sci. USA, 75 1929 (1978)) may also be
applied. In addition, a coryneform bacterium may be transformed by
the electric pulse method (Sugimoto et al., JP02-207791A).
[0091] Examples of a temperature-sensitive plasmid for a coryneform
bacterium include p48K and pSFKT2 (JP2000-262288A), and pHSC4
(France Patent No. 2667875 (1992) and JP05-7491A). These plasmids
are autonomously replicable in a coryneform bacterium at least at
25.degree. C., but they are not autonomously replicable at
37.degree. C. Escherichia coli AJ12571 having pHSC4 has been
deposited with an Accession No. FERM P-11763 at National Institute
of Bioscience and Human-Technology, Agency of industrial Science
and Technology, Ministry of International Trade and Industry
(currently, International Patent Organism Depositary, National
Institute of Advanced Industrial Science and Technology) (Central
6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-5466 Japan) on Oct. 11,
1990, and then transferred to an international deposit under the
provisions of Budapest Treaty on Aug. 26, 1991 with an Accession
No. FERM BP-3524.
[0092] A transformant obtained as described above is cultured at a
temperature at which the temperature-sensitive replication origin
does not function (25.degree. C.), to thereby obtain a strain into
which the plasmid has been introduced. The plasmid-introduced
strain is cultured at high temperature to excise the
temperature-sensitive plasmid, and the bacterial strain is applied
onto a plate containing an antibiotic. The temperature-sensitive
plasmid cannot replicate at high temperature. Therefore, a
bacterial strain from which the plasmid has been excised cannot
grow on a plate containing an antibiotic, but a bacterial strain in
which recombination has occurred between the ach gene on the
plasmid and the ach gene on a chromosome appears at a very low
frequency.
[0093] In the strain obtained by introducing the recombinant DNA
into a chromosome as described above, recombination occurs with an
ach gene sequence that is originally present on a chromosome, and
two fusion genes of the chromosomal ach gene and the deleted-form
of ach gene are inserted into a chromosome so that other portions
of the recombinant DNA (vector part, temperature-sensitive
replication origin and drug-resistance marker) are present between
the fusion genes.
[0094] Then, in order to leave only the deleted-form of ach gene on
a chromosomal DNA, the gene is eliminated together with the vector
portion (the temperature-sensitive replication origin and
drug-resistance marker) from the chromosomal. This procedure causes
a case where the normal ach gene remains on the chromosomal DNA and
the deleted-form of ach gene is excised, or to the contrary, a case
where the normal ach gene is excised and the deleted-form of ach
gene remains on chromosomal DNA. In both cases, when culture is
performed at a temperature that allows a temperature-sensitive
replication origin to function, the cleaved DNA is kept in a cell
as a plasmid. Next, when culture is performed at a temperature that
does not allow a temperature-sensitive replication origin to
function, the ach gene on the plasmid is eliminated from the cell
together with the plasmid. Then, a strain in which the deleted-form
of ach gene remains on the chromosome, is selected by PCR, Southern
hybridization, or the like, to thereby yield a strain in which the
ach gene is disrupted.
[0095] In the case where a plasmid having no replicability in a
coryneform bacterium is used instead of the above-mentioned
temperature-sensitive plasmid, gene disruption can also be
performed in a similar way. The plasmid having no replicability in
a coryneform bacterium is preferably a plasmid having a
replicability in Escherichia coli, and examples thereof include
pHSG299 (Takara Bio Inc.) and pHSG399 (Takara Bio Inc.).
[0096] Meanwhile, examples of a method of decreasing an activity of
acetyl-CoA hydrolase include not only the above-mentioned genetic
engineering method but also a method comprising treating a
coryneform bacterium with ultraviolet irradiation or with a
mutagenesis agent to be generally used for mutation such as
N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid, and
selecting a bacterial strain having decreased acetyl-CoA hydrolase
activity.
[0097] In the present invention, it is more effective to use a
bacterial strain modified so that either or both of activities of
phosphotransacetylase (hereinafter, referred to as PTA) and acetate
kinase (hereinafter, referred to as ACK) are decreased in addition
to the decrease in the ACH activity.
[0098] In the present invention, phosphotransacetylase (PTA)
activity means an activity to catalyze a reaction to generate
acetyl phosphate by transferring phosphate to acetyl-CoA
(EC:2.3.1.8), and acetate kinase (ACK) means an activity to
catalyze a reaction to generate acetic acid from acetyl phosphate
and ADP (EC:2.7.2.1).
[0099] Decreasing these activities may be accomplished by
disruption of genes encoding the above-mentioned enzymes, or by
modification of an expression regulatory sequence such as a
promoter and Shine-Dalgarno (SD) sequence of the genes encoding the
enzymes, Gene disruption can be performed in the same way as the
above-mentioned method of disrupting the ach gene.
[0100] As genes encoding the enzymes, for example, the following
genes of Corynebacterium glutamicum deposited in GenBank may be
used:
[0101] pta (phosphoacetyltransferase) gene: GenBank Accession No.
NCg12657 (complementary strand of nucleotide numbers
2936506-2937495 in NC.sub.--003450) (the nucleotide numbers
956-1942 in SEQ ID NO: 39) ack (acetate kinase) gene: GenBank
Accession No. NCg12656 (complementary strand of nucleotide numbers
2935313-2936506 in NC.sub.--003450) (the nucleotide numbers
1945-3135 in SEQ ID NO: 39).
[0102] The phrase "phosphotransacetylase (hereinafter, referred to
as PTA) activity is decreased" means that PTA activity is decreased
as compared to a PTA-unmodified strain. The PTA activity is
decreased than PTA-unmodified strain or a wild-type strain, and it
is preferably decreased to 50% or less per bacterial cell, more
preferably 10% or less per bacterial cell. The PTA activity may be
completely eliminated. The decrease in PTA activity can be
confirmed by determining the PTA activity by a method of Klotzsch
et al. (Klotzsch H. R., Meth Enzymol. 12, 381-386 (1969)). A
coryneform bacterium in which activities of both of ACH and PTA are
decreased can be obtained by constructing a coryneform bacterium
having decreased ACH activity and modifying it so as to decrease
PTA activity. However, there is no preference in the order for
performing the modification to decrease PTA activity and the
modification to decrease ACH activity.
[0103] The phrase "acetate kinase (hereinafter, referred to as ACK)
activity is decreased" means that ACK activity is decreased as
compared to a wild-type strain or an ACK-unmodified strain. The ACK
activity is decreased than ACK-unmodified strain or a wild-type
strain, and it is preferably decreased to 50% or less per bacterial
cell, more preferably 10% or less per bacterial cell as compared to
an ACK-unmodified strain. ACK activity may be completely
eliminated. The decrease in ACK activity can be confirmed by
determining the ACK activity by a method of Ramponi et al. (Ramponi
G., Meth. Enzymol. 42, 409-426 (1975)). A coryneform bacterium in
which activities of both of ACH and ACK are decreased can be
obtained by constructing a coryneform bacterium having decreased
ACH activity and modifying it so as to decrease ACK activity.
However, there is no preference in the order for performing the
modification to decrease ACK activity and the modification to
decrease ACH activity.
[0104] In the present invention, it is more effective to use a
bacterial strain modified so that a lactate dehydrogenase
(hereinafter, referred to as LDH) activity is decreased in addition
to decrease in the above-mentioned ACH activity. The lactate
dehydrogenase activity means an activity to catalyze a reaction to
generate lactic acid by reducing pyruvic acid using NADH as a
coenzyme. The phrase "lactate dehydrogenase activity is decreased"
means that LDH activity is decreased as compared to an
LDH-unmodified strain. The LDH activity is decreased than an
LDH-unmodified strain or a wild-type strain, and it is preferably
decreased to 50% or less per bacterial cell, preferably 10% or less
per bacterial cell. LDH activity may be completely eliminated. The
decrease in LDH activity can be confirmed by determining the LDH
activity by a method of L. Kanarek et al. (L. Kanarek and R. L.
Hill, J. Biol. Chem. 239, 4202 (1964)). A coryneform bacterium of
the present invention can be obtained by preparing a coryneform
bacterium having decreased LDH activity and modifying it so as to
decrease ACH activity. However, there is no preference in the order
for performing the modification to decrease LDH activity and the
modification to decrease ACH activity.
[0105] As an ldh gene, there may be used, for example, a gene
having a sequence represented by SEQ ID NO: 37, and gene disruption
may be performed in a similar manner as in the case of the
above-mentioned ach gene.
[0106] Furthermore, in the present invention, there may be used a
bacterium modified so that an activity of pyruvate carboxylase
(hereinafter, referred to as PC) is increased in addition to
decrease in ACH activity. The term "pyruvate carboxylase activity
is increased" means that PC activity is increased as compared to a
wild-type strain or an unmodified strain such as a parent strain.
PC activity can be determined by a method of Peters-Wendisch P. G
et al. (Peters-Wendisch P. G. et al. Microbiology 143, 1095-1103
(1997)).
[0107] As a PC gene encoding a PC protein to be used in the method
of the present invention, there may be employed a gene whose
nucleotide sequence has been determined, or a gene obtained by
isolating a DNA fragment that encodes a protein having PC activity
from a chromosome of microorganisms, animals, plants, or the like
according to the method described below and determining its
nucleotide sequence. Further, after determination of the nucleotide
sequence, a gene synthesized based on the sequence may be used. For
example, there may be used a pyruvate carboxylase gene of
Corynebacterium glutamicum ATCC13032 (GenBank Accession No.
NCg10659 gene: SEQ ID NO: 46). Further, there may also be used PC
genes derived from the following organisms.
[0108] Human [Biochem. Biophys. Res. Comm., 202, 1009-1014,
(1994)]
[0109] Mouse [Proc. Natl. Acad. Sci. USA., 90, 1766-1779,
(1993)]
[0110] Rat [GENE, 165, 331-332, (1995)]
[0111] Yeast; Saccharomyces cerevisiae [Mol. Gen. Genet., 229,
307-315, (1991)]Schizosaccharomyces pombe [DDBJ Accession No.;
D78170]
[0112] Bacillus stearothermophilus [GENE, 191, 47-50, (1997)]
[0113] Rhizobium etli [J. Bacteriol., 178, 5960-5970, (1996)]
[0114] A DNA fragment containing a PC gene can be expressed by
inserting the DNA fragment into a suitable expression plasmid such
as pUC118 (Takara Bio Inc.), and introducing into a suitable host
microorganism such as Escherichia coli JM109 (Takara Bio Inc.). The
expressed PC gene product, which is pyruvate carboxylase, can be
confirmed by determining PC activity by the known method as
described above in the transformant, and then comparing the
determined PC activity with PC activity of a crude enzyme solution
extracted from a non-transformant strain. The DNA fragment
containing PC gene is inserted into a suitable plasmid such as a
plasmid vector containing at least a gene responsible for
replication function of the plasmid in coryneform bacteria, thereby
a recombinant plasmid capable of highly expressing PC in coryneform
bacteria can be obtained. Here, in the recombinant plasmid, a
promoter for expression of PC gene may be a promoter of coryneform
bacteria. However, it is not limited to such a promoter; and any
promoter can be used as long as it has a nucleotide sequence
capable of initiating transcription of PC gene.
[0115] Plasmid vectors, into which PC gene can be introduced, are
not limited as long as they contain at least a gene responsible for
replication function in coryneform bacteria. Specific examples
thereof include: plasmid pCRY30 described in JP03-210184A; plasmids
pCRY2I, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX each
described in JP02-72876A and U.S. Pat. No. 5,185,262; plasmids
pCRY2 and pCRY3 each described in JP01-191686A; pAM330 described in
JP58-67679A; pHM1519 described in JP58-77895A; pAJ655, pAJ611, and
pAJ1844 each described in JP58-192900A; pCG1 described in
JP57-134500A; pCG2 described in JP58-35197A; and pCGG4 and pCG11
each described in JP57-1 83799A.
[0116] Of those, plasmid vectors used in host-vector system for
coryneform bacteria are preferably those having a gene responsible
for replication function of the plasmid in coryneform bacteria and
a gene responsible for stabilization function of the plasmid in
coryneform bacteria. For instance, plasmids pCRY30, pCRY2I,
pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX can be preferably
used.
[0117] Coryneform bacteria having enhanced PC gene expression can
be obtained by transforming a coryneform bacterium, for example,
Brevibacterium lactofermentum 2256 strain (ATCC13869) with a
recombinant vector prepared by inserting PC gene into an
appropriate site of a plasmid vector which is replicable in aerobic
coryneform bacteria as described above. Transformation can be
carried out by, for example, the electric pulse method (Res.
Microbiol., Vol. 144, p. 181-185, 1993). PC activity can also be
increased by enhancing gene expression by introduction,
substitution, amplification or the like of PC gene on a chromosome
by a known homologous recombination method. By disrupting the ach
gene in a strain which highly expresses the PC gene, a bacterial
strain with enhanced PC activity and decreased ACH activity can be
obtained. There is no preference in the order for performing
modifications to decrease ACH activity and to enhance PC
activity.
[0118] Moreover, in the present invention, a bacterium that has
been modified so that activity of ACH, or activities of ACH, PTA
and ACK is/are decreased, and further modified so that LDH activity
is decreased and PC activity is increased, is particularly
effectively used for production of a substance, especially for
production of succinic acid.
<3> Production of Succinic Acid Using the Bacterium of the
Present Invention
[0119] Succinic acid can be efficiently produced by culturing the
thus obtained coryneform bacterium in a medium to produce and
accumulate succinic acid in the medium and collecting succinic acid
from the medium.
[0120] Upon use of the above-mentioned bacterium in reaction for
producing succinic acid, the bacterium subjected to slant culture
on such a solid medium as an agar medium may be used directly for
the reaction, but a bacterium obtained by culturing the
above-mentioned bacterium in a liquid medium (seed culture) in
advance may be preferably used. Succinic acid may be produced by
allowing the seed-cultured bacterium to react with an organic
material while the bacterium is proliferating in a medium
containing the organic raw material. In addition, succinic acid can
also be produced by harvesting bacterial cells which has been
proliferated and then reacting the bacterial cells with an organic
raw material in reaction liquid containing the organic raw
material. Further, for the purpose of using an aerobic coryneform
bacterium in the method of the present invention, it is preferable
to use the aerobic coryneform bacterium for the reaction after
culturing the bacterium under a normal aerobic condition. The
medium to be used for culture may be any medium normally used for
culturing microorganisms. For instance, conventional media, which
can be prepared by adding natural nutrient sources such as meat
extract, yeast extract and peptone to a composition made of
inorganic salts such as ammonium sulfate, potassium phosphate and
magnesium sulfate, can be used. In the case of harvesting and using
the bacterial cells after culture, the bacterial cells are
harvested by centrifugation, membrane separation, or the like, and
then used for the reaction.
[0121] In the present invention, a treated product of bacterial
cells can also be used. For instance, the treated products of
bacterial cells include immobilized bacterial cells which are
immobilized on acrylamide, carrageenan or the like, disrupted
bacterial cells, centrifugal supernatant thereof, or fractions
obtained by partially purifying the supernatant with an ammonium
sulfate treatment or the like.
[0122] An organic raw material to be used for the production method
of the present invention is not particularly limited as long as it
is a carbon source which can be assimilated by the microorganism
described herein to produce succinic acid. In general, there is
used a fermentable carbohydrate including: a carbohydrate such as
galactose, lactose, glucose, fructose, glycerol, sucrose,
saccharose, starch and cellulose; polyalcohol such as glycerin,
mannitol, xylitol and ribitol. Of those, glucose, fructose and
glycerol are preferable, and glucose is particularly
preferable.
[0123] In addition, a saccharified starch liquid, molasses and the
like, which contain any one of the above-mentioned fermentable
carbohydrates, can also be used. Any one of those fermentable
carbohydrates may be used alone or may be used in combination. The
concentration at which the above-mentioned organic raw material is
used is not particularly limited, but it is advantageous to
increase the concentration as high as possible within the range
that does not inhibit the production of succinic acid. The reaction
is generally performed under the presence of the organic raw
material in the range of 5 to 30% (w/v), preferably 10 to 20%
(w/v). The organic raw material may be additionally added according
to a decrease in the above-mentioned organic raw material when the
reaction progresses.
[0124] The reaction liquid containing the organic raw material is
not particularly limited. The reaction liquid to be used may be
water, buffer, medium or the like, but the medium is most
preferable. The reaction liquid is preferably one containing a
nitrogen source, inorganic salts and the like. Here, the nitrogen
source is not particularly limited as long as it can be assimilated
by the microorganism described herein to produce succinic acid.
Specific examples of the nitrogen source include various organic
and inorganic nitrogen compounds such as ammonium salts, nitrate,
urea, soybean hydrolysate, casein hydrolysate, peptone, yeast
extract, meat extract, and corn steep liquor. Examples of the
inorganic salts include various kinds of phosphoric acid salts,
sulfuric acid salts and metal salts of magnesium, potassium,
manganese, iron, zinc, and the like. In addition, components that
promote growth of bacterial cells including: vitamins such as
biotin, pantothenic acid, inositol and nicotinic acid; nucleotides;
and amino acids, may be added if necessary. Further, it is
preferable that an appropriate amount of a commercially available
antifoaming agent is added to the reaction liquid to suppress
foaming at the time of reaction.
[0125] pH of the reaction liquid can be adjusted by adding sodium
carbonate, sodium bicarbonate, potassium carbonate, potassium
bicarbonate, magnesium carbonate, sodium hydroxide, calcium
hydroxide, magnesium hydroxide, or the like. The pH for the
reaction is usually 5 to 10, preferably 6 to 9.5, so the pH of the
reaction liquid is adjusted within the above-mentioned range with
an alkaline material, carbonate, urea, or the like during the
reaction, if necessary.
[0126] The medium preferably contains carbonate ion, bicarbonate
ion or carbonic acid gas (carbon dioxide). The carbonate ion or
bicarbonate ion is supplied from magnesium carbonate, sodium
carbonate, sodium bicarbonate, potassium carbonate, or potassium
bicarbonate, which can also be used as a neutralizing agent.
However, if necessary, the carbonate ion or bicarbonate ion can
also be supplied from carbonic acid or bicarbonic acid, or salts
thereof, or carbonic acid gas. Specific examples of the salts of
carbonic acid or bicarbonic acid include magnesium carbonate,
ammonium carbonate, sodium carbonate, potassium carbonate, ammonium
bicarbonate, sodium bicarbonate, and potassium bicarbonate. In
addition, the carbonate ion or bicarbonate ion is added at a
concentration of 0.001 to 5 M, preferably 0.1 to 3 M, and more
preferably 1 to 2 M. When carbonic acid gas is contained, the
amount of the carbonic acid gas to be contained is 50 mg to 25 g,
preferably 100 mg to 15 g, and more preferably 150 mg to 10 g per
liter of the liquid.
[0127] The optimal temperature at which the bacterium to be used in
the reaction grow is generally in the range of 25.degree. C. to
35.degree. C. On the other hand, the temperature at the time of
reaction is generally in the range of 25.degree. C. to 40.degree.
C., preferably in the range of 30.degree. C. to 37.degree. C. The
amount of bacterial cells to be used in the reaction is not
particularly limited, but the amount is adjusted in the range of 1
to 700 g/L, preferably 10 to 500 g/L, and more preferably 20 to 400
g/L. The time period of the reaction is preferably 1 to 168 hours,
more preferably 3 to 72 hours.
[0128] Upon culture of the bacterium, it is necessary to supply
oxygen by aeration and agitation. On the other hand, the reaction
for producing succinic acid may be performed with aeration and
agitation, or may be performed under an anaerobic condition with
neither aeration nor supply of oxygen. Here, the term "anaerobic
condition" means that the reaction is conducted while keeping the
dissolved oxygen low in the liquid. In this case, it is preferable
to carry out the reaction at a dissolved oxygen of 0 to 2 ppm,
preferably 0 to 1 ppm, and more preferably 0 to 0.5 ppm. For that
purpose, there may be used a method in which a vessel is
hermetically sealed to carry out the reaction without aeration; a
method in which an inert gas such as a nitrogen gas is supplied to
carry out the reaction; a method in which aeration with an inert
gas containing carbonic acid gas is performed; and the like.
[0129] Succinic acid accumulated in the reaction liquid (culture
solution) can be isolated and purified from the reaction liquid
according to a conventional procedure. To be specific, succinic
acid can be isolated and purified by removing solid materials
including bacterial cells through centrifugation, filtration or the
like, and desalting the solution with an ion exchange resin or the
like, followed by crystallization or column chromatography from the
solution.
[0130] In the present invention, after production of succinic acid
by the method of the present invention as described above, a
polymerization reaction is carried out using the obtained succinic
acid as a raw material to produce a succinic acid-containing
polymer. The succinic acid-containing polymer may be a homopolymer
or a copolymer with other polymer raw materials. In recent years,
environment-friendly industrial products are on the increase, and
polymers prepared by using raw materials of a plant origin have
been attracting attention. The succinic acid to be produced in the
present invention can be processed into polymers such as polyester
and polyamide and then used. Specific examples of the succinic
acid-containing polymer include a succinic acid-containing
polyester obtained through polymerization between a diol such as
butanediol or ethylene glycol and succinic acid, and a succinic
acid-containing polyamide obtained through polymerization between a
diamine such as hexamethylenediamine and succinic acid.
[0131] Further, succinic acid or a composition containing succinic
acid which can be obtained by the production method of the present
invention can be used as food additives, pharmaceuticals,
cosmetics, and the like.
EXAMPLES
[0132] Hereinafter, the present invention will be described in
further detail with reference to examples.
Example 1
<1> Construction of A Disruption Vector Carrying sacB
Gene
(A) Construction of pBS3
[0133] The sacB gene (SEQ ID NO: 35) was obtained by PCR using a
chromosomal DNA of Bacillus subtilis as a template and SEQ ID NOS:
1 and 2 as primers. PCR was carried out using LA Taq (Takara Bio
Inc.) in such a way that one cycle of heat-retention at 94.degree.
C. for 5 minutes was performed and then a cycle of denaturation at
94.degree. C. for 30 seconds, annealing at 49.degree. C. for 30
seconds and elongation at 72.degree. C. for 2 minutes was repeated
25 times. The PCR product thus obtained was purified by a
conventional procedure and then digested with BglII and BamHI,
followed by blunt-ending. The fragment was inserted into a site of
pHSG299 which had been digested with AvaII and blunt-ended.
Competent cells of Escherichia coli JM109 (Takara Bio Inc.) were
used for transformation with this DNA and the transformed cells
were applied on an LB medium containing 25 .mu.g/ml kanamycin
(hereinafter, abbreviated as Km), followed by overnight culture.
Subsequently, appeared colonies were picked up, and single colonies
were isolated, thereby transformants were obtained. Plasmids were
extracted from transformants, and a plasmid into which a PCR
product of interest was inserted was named pBS3. FIG. 1 shows the
construction procedures of pBS3.
(B) Construction of pBS4S
[0134] SmaI site in the kanamycin resistance gene sequence present
on pBS3 was disrupted by crossover PCR-mediated nucleotide
substitution causing no amino acid substitution to obtain a
plasmid. First, PCR was carried out using pBS3 as a template and
synthetic DNAs of SEQ ID NOS: 3 and 4 as primers, thereby amplified
product of N-terminal region of the kanamycin resistance gene was
obtained. On the other hand, to obtain amplified product of
C-terminal region of the Km resistance gene, PCR was carried out
using pBS3 as a template and synthetic DNAs of SEQ ID NOS: 5 and 6
as primers. The PCR was carried out using Pyrobest DNA Polymerase
(Takara Bio Inc.) in such a way that one cycle of heat-retention at
98.degree. C. for 5 minutes was performed and then a cycle of
denaturation at 98.degree. C. for 10 seconds, annealing at
57.degree. C. for 30 seconds and elongation at 72.degree. C. for 1
minute was repeated 25 times, to thereby yield a PCR product of
interest. SEQ ID NOS: 4 and 5 are partially complementary to each
other, and the SmaI site in the sequence is disrupted by nucleotide
substitution causing no amino acid substitution. Next, to obtain a
fragment of a mutant kanamycin resistance gene in which the SmaI
site is disrupted, the gene products of the N-terminal and
C-terminal regions of the above-mentioned kanamycin resistance gene
were mixed at an approximately equimolar concentration, and PCR was
carried out using the mixture of the gene products as templates and
synthetic DNAs of SEQ ID NOS: 3 and 6 as primers, to thereby yield
amplified product of a mutation-introduced Km resistance gene. PCR
was carried out using Pyrobest DNA Polymerase (Takara Bio Inc.) in
such a way that one cycle of heat-retention at 98.degree. C. for 5
minutes was performed and then a cycle of denaturation at
98.degree. C. for 10 seconds, annealing at 57.degree. C. for 30
seconds and elongation at 72.degree. C. for 1.5 minutes was
repeated 25 times, to thereby yield a PCR product of interest.
[0135] The PCR product was purified by a conventional procedure and
then digested with BanII, followed by insertion into BanII site of
the above-mentioned pBS3. Competent cells of Escherichia coli JM109
(Takara Bio Inc.) were used for transformation with this DNA and
transformed cells were applied on an LB medium containing 25
.mu.g/ml of kanamycin, followed by overnight culture. Subsequently,
appeared colonies were picked up, and single colonies were
isolated, thereby transformants were obtained. Plasmids were
extracted from the transformants, and a plasmid into which a PCR
product of interest was inserted was named pBS4S. FIG. 2 shows the
construction procedures of pBS4S.
(C) Construction of pBS5T
[0136] A plasmid was constructed by inserting a
temperature-sensitive replication origin for a coryneform bacterium
into pBS4S constructed in the above-mentioned (B). That is, a
temperature-sensitive replication origin for a coryneform bacterium
was obtained by digesting pHSC4 (JP05-7491 A) with BamHI and SmaI,
followed by blunt-ending, and the temperature-sensitive replication
origin was inserted into a blunt-ended Ndel site of pBS4S.
Competent cells of Escherichia coli JM109 (Takara Bio Inc.) were
used for transformation with this DNA and transformed cells were
applied on an LB medium containing 25 .mu.g/ml of Km, followed by
overnight culture. Subsequently, appeared colonies were picked up,
and single colonies were isolated, thereby transformants were
obtained. Plasmids were extracted from the transformants, and a
plasmid into which a PCR product of interest was inserted was named
pBS5T. FIG. 3 shows the construction procedures of pBS5T.
Example 2
Construction of LDH Gene-Disrupted Strain
(A) Cloning of A Fragment For Disrupting Lactate Dehydrogenase
Gene
[0137] A fragment of a lactate dehydrogenase gene (hereinafter;
abbreviated as ldh gene) derived from Brevibacterium lactofermentum
2256 strain in which ORF thereof was deleted was obtained by
crossover PCR using as primers synthetic DNAs designed based on the
nucleotide sequence (SEQ ID NO: 37) of the gene of Corynebacterium
glutamicum ATCC13032 (GenBank Database Accession No.
NC.sub.--003450), which has already been disclosed. That is, PCR
was carried out by a conventional procedure using a chromosomal DNA
of Brevibacterium lactofermentum 2256 strain as a template and
synthetic DNAs of SEQ ID NOS: 7 and 8 as primers, thereby amplified
product of the N-terminal region of the ldh gene was obtained. On
the other hand, to obtain amplified product of the C-terminal
region of the ldh gene, PCR was carried out by a conventional
procedure using a genomic DNA of Brevibacterium lactofermentum 2256
as a template and synthetic DNAs of SEQ ID NOS: 9 and 10 as
primers. SEQ ID NO: 8 and 9 are complementary to each other and
have structures for deleting the entire sequences of ldh ORF.
[0138] Brevibacterium lactofermentum 2256 strain is available from
the American Type Culture Collection (ATCC) (Address: ATCC, P.O.
Box 1549, Manassas, Va. 20108, United States of America).
[0139] Next, to obtain a fragment of the ldh gene in which its
internal sequence is deleted, the above-mentioned gene products of
the N-terminal and C-terminal regions of ldh were mixed at an
approximately equimolar concentration, and PCR was carried out by a
conventional procedure using the mixture of the gene products as
templates and synthetic DNAs of SEQ ID NOS: 11 and 12 as primers,
to thereby yield amplified product of the mutation-introduced ldh
gene. The PCR product thus obtained was purified by a conventional
procedure and then digested with SalI, followed by insertion into
SalI site of the above-mentioned pBS4S. Competent cells of
Escherichia coli JM109 (Takara Bio Inc.) were used for
transformation with this DNA and transformed cells were applied on
an LB medium containing 100 .mu.M of IPTG, 40 .mu.g/ml of X-Gal,
and 25 .mu.g/ml of Km, followed by overnight culture, Subsequently,
appeared white colonies were picked up, and single colonies were
isolated, thereby transformants were obtained. Plasmids were
extracted from the transformants, and a plasmid into which a PCR
product of interest was inserted was named p.DELTA.ldh56-1. FIG. 4
shows the construction procedures of the plasmid.
(B) Preparation of ldh-Disrupted Strain
[0140] The p.DELTA.ldh56-1 obtained by the above-mentioned (A) does
not contain a region that enables autonomous replication in a cell
of a coryneform bacterium. Therefore, when a coryneform bacterium
is transformed with this plasmid, a strain in which the plasmid is
integrated into a chromosome by homologous recombination appears at
a very low frequency as a transformant. Brevibacterium
lactofermentum 2256 strain was transformed using a high
concentration of the plasmid p.DELTA.ldh56-1 by the electric pulse
method, and the transformed cells were applied on CM-Dex medium (5
g/L of glucose, 10 g/L of polypeptone, 10 g/L of yeast extract, 1
g/L of KB.sub.2PO.sub.4, 0.4 g/L of MgSO.sub.4.7H.sub.2O, 0.01 g/L
of FeSO.sub.4.7H.sub.2O, 0.01 g/L of MnSO.sub.4.7H.sub.2O, 3 g/L of
urea, 1.2 g/L of soybean hydrolysate, pH 7.5 (KOH)) containing 25
.mu.g/ml of kanamycin, followed by culture at 31.5.degree. C. for
about 30 hours. A strain grown on the medium contains the kanamycin
resistance gene and sacB gene which are derived from the plasmid on
the genome, as a result of homologous recombination between the ldh
gene fragment on the plasmid and the ldh gene on a genome of
Brevibacterium lactofermentum 2256 strain.
[0141] Next, the single cross-over recombinant was cultured at
31.5.degree. C. overnight in CM-Dex liquid medium not containing
kanamycin, and after suitable dilution, it was applied on 10%
sucrose-containing Dex-S10 medium (100 g/L of sucrose, 10 g/L of
polypeptone, 10 g/L of yeast extract, 1 g/L of KH.sub.2PO.sub.4,
0.4 g/L of MgSO.sub.4.7H.sub.2O, 0.01 g/L of FeSO.sub.4.7H.sub.2O,
0.01 g/L of MnSO.sub.4.4H.sub.2O, 3 g/L of urea, 1.2 g/L of soybean
hydrolysate, 10 .mu.g/L of biotin, pH 7,5 (KOH)) not containing
kanamycin, followed by culture at 31.5.degree. C. for about 30
hours. As a result, about 50 strains, which were considered to
become sucrose-insensitive due to elimination of the sacB gene by
second homologous recombination, were obtained.
[0142] The strains thus obtained include: a strain in which ldh
gene was replaced by the mutant type derived from p.DELTA.ldh56-1;
and a strain in which ldh gene reverted to the wild type. Whether
the ldh gene is the mutant type or the wild type can be confirmed
easily by directly subjecting the bacterial strains obtained by
culturing on Dex-S10 agar medium to PCR and detecting their ldh
gene. In PCR analysis using primers (SEQ ID NOS: 7 and 10) for
amplifying ldh gene, a strain which yielded a PCR product having a
smaller size than that of a product obtained by PCR using a
chromosomal DNA of the 2256 strain as a template was defined as an
ldh-disrupted strain and used in the following experiments. As a
result of the analysis of the sucrose-insensitive strains by the
above-mentioned method, a strain carrying only the mutant type gene
was selected and named 2256.DELTA.(ldh) strain. Also, the strain
was used as a parent strain of the following acetic
acid-biosynthetic gene-disrupted strain.
Example 3
Construction of Acetate Kinase Gene-Disrupted Strain
(A) Cloning of A Fragment For Disrupting Acetate Kinase Gene
[0143] A fragment of an acetate kinase gene (hereinafter,
abbreviated as ack) of Brevibacterium lactofermentum 2256 strain in
which ORF thereof was deleted was obtained by crossover PCR using
as primers synthetic DNAs designed based on the nucleotide sequence
(1945-3135 in SEQ ID NO: 39) of the gene of Corynebacterium
glutamicum ATCC13032 (GenBank Database Accession No.
NC.sub.--003450), which has already been disclosed. That is, PCR
was carried out using a genomic DNA of Brevibacterium
lactofermentum 2256 strain as a template and synthetic DNAs of SEQ
ID NOS: 13 and 14 as primers, thereby amplified product of
N-terminal region of the ack gene was obtained. On the other hand,
to obtain amplified product of C-terminal region of the ack gene,
PCR was carried out using a genomic DNA of Brevibacterium
lactofermentum 2256 as a template and synthetic DNAs of SEQ ID NOS:
15 and 16 as primers. SEQ ID NO: 14 and 15 are complementary to
each other. The PCR was carried out using KOD-plus-(TOYOBO) in such
a way that one cycle of heat-retention at 94.degree. C. for 2
minutes was performed and then, for the N-terminal region, a cycle
of denaturation at 94.degree. C. for 10 seconds, annealing at
55.degree. C. for 30 seconds and elongation at 68.degree. C. for 30
seconds, and for the C-terminal region, a cycle of denaturation at
94.degree. C. for 10 seconds, annealing at 55.degree. C. for 30
seconds and elongation at 68.degree. C. for 2 minutes, were
repeated 30 times, respectively. Next to obtain a fragment of the
ack gene in which its internal sequence is deleted, the
above-mentioned gene products of the N-terminal and C-terminal
regions of ack were mixed at an approximately equimolar
concentration, and PCR was carried out using the mixture of the
gene products as templates and synthetic DNAs of SEQ ID NOS: 17 and
18 as primers, to thereby yield amplified product of the
mutation-introduced ack gene. The PCR was carried out using
KOD-plus-(TOYOBO) in such a way that one cycle of heat-retention at
94.degree. C. for 2 minutes was performed and then a cycle of
denaturation at 94.degree. C. for 10 seconds, annealing at
55.degree. C. for 30 seconds and elongation at 68.degree. C. for
2.5 minutes was repeated 30 times, to thereby yield an amplified
product of the mutation-introduced ack gene of interest.
[0144] The PCR product thus obtained was purified by a conventional
procedure and then digested with XbaI, followed by insertion into
XbaI site in pBS5T constructed in the above-mentioned Example 1
(C). Competent cells of Escherichia coli JM109 (Takara Bio Inc.)
were used for transformation with this DNA and transformed cells
were applied on an LB medium containing 100 .mu.M of IPTG, 40
.mu.g/ml of X-Gal, and 25 .mu.g/ml of kanamycin, followed by
overnight culture. Subsequently, appeared white colonies were
picked up, and single colonies were isolated, thereby transformants
were obtained. Plasmids were extracted from the transformants, and
a plasmid into which a PCR product of interest was inserted was
named pBS5T::.DELTA.ack. FIG. 5 shows the construction procedures
of pBS5T::.DELTA.ack.
(B) Preparation of ack-Disrupted Strain
[0145] The replication origin for coryneform bacteria in
pBS5T::.DELTA.ack obtained by the above-mentioned (A) is
temperature-sensitive. That is, the plasmid is autonomously
replicable in a cell of a coryneform bacterium at 25.degree. C.,
but it is not autonomously replicable at 31.5.degree. C. (or
34.degree. C.). Brevibacterium lactofermentum 2256.thrfore.(ldh)
strain was transformed using the plasmid by the electric pulse
method, and transformed cells were applied on a CM-Dex medium
containing 25 .mu.g/ml of kanamycin, followed by culture at
25.degree. C. for 2 nights. Appeared colonies were isolated, to
thereby yield transformants. The transformants contain the plasmid.
The transformants were cultured at 34.degree. C. overnight in a
CM-Dex medium (5 g/L of glucose, 10 g/L of polypeptone, 10 g/L of
yeast extract, 1 g/L of KH.sub.2PO.sub.4, 0.4 g/L of
MgSO.sub.4.7H.sub.2O, 0.01 g/L of FeSO.sub.4.7H.sub.2O, 0.01 g/L of
MnSO.sub.4.7H.sub.2O, 3 g/L of urea, 1.2 g/L of soybean
hydrolysate, pH 7.5 (KOH)) not containing kanamycin and after
suitable dilution, it was applied on a CM-Dex medium containing 25
.mu.g/ml of kanamycin, followed by culture at 34.degree. C. for
about 30 hours, The strain grown on the medium contains the
kanamycin resistance gene and sacB gene which are derived from the
plasmid on the genome, as a result of homologous recombination
between the ack gene fragment on the plasmid and the ack gene on
the genome of Brevibacterium lactofermentum 2256.DELTA.(ldh)
strain.
[0146] Next, the singe crossover recombinant was cultured at
31.5.degree. C. overnight in CM-Dex liquid medium not containing
kanamycin, and after suitable dilution, it was applied on 10%
sucrose-containing Dex-S10 medium not containing kanamycin,
followed by culture at 31.5.degree. C. for about 30 hours. As a
result, about 50 strains which were considered to become
sucrose-insensitive due to elimination of the sacB gene by the
second homologous recombination, were obtained.
[0147] The thus obtained strains include: a strain in which ack
gene was replaced by the mutant type derived from
pBS5T::.DELTA.ack; and a strain in which ack gene reverted to the
wild type. Whether ack gene is the mutant type or the wild type can
be confirmed easily by directly subjecting a bacterial strain
obtained through culturing on a Dex-S10 agar medium to PCR and
detecting the ack gene. Analysis of the ack gene by using primers
(SEQ ID NOS: 13 and 16) for PCR amplification should result in a
DNA fragment of 3.7 kb for the wild type and a DNA fragment of 2.5
kb for the mutant type having a deleted region. As a result of the
analysis of the sucrose-insensitive strain by the above-mentioned
method, a strain carrying only the mutant type gene was selected
and named 2256.DELTA.(ldh, ack).
Example 4
Construction of A Strain In Which Acetate Kinase Gene And
Phosphotransacetylase Gene Are Disrupted
(A) Cloning of Fragments For Disrupting Acetate Kinase Gene And
Phosphotransacetylase Gene
[0148] The ORFs of acetate kinase gene ("ack") and
phosphotransacetylase gene (hereinafter, referred to as pta) of
Brevibacterium lactofermentum 2256 strain have an operon structure,
and both ORFs can be made deficient simultaneously. These gene
fragments were obtained by cross-over PCR using as primers
synthetic DNAs designed based on the nucleotide sequences of the
genes of Corynebacterium glutamicum ATCC13032 (GenBank Database
Accession No. NC.sub.--003450) (pta-ack gene; SEQ ID NO: 39). That
is, PCR was carried out using a genomic DNA of Brevibacterium
lactofermentum 2256 strain as a template, and synthetic DNAs of SEQ
ID NOS: 19 and 20 as primers, to thereby yield an amplified product
of N-terminal region of the pta gene. On the other hand, to yield
an amplified product of C-terminal region of the ack gene, PCR was
carried out using a genomic DNA of Brevibacterium lactofermentum
2256 strain as a template and synthetic DNAs of SEQ ID NOS: 21 and
16 as primers. SEQ ID NOS: 20 and 21 are partially complementary to
each other. PCR was performed by using KOD-plus-(TOYOBO) in such a
way that one cycle of heat-retention at 94.degree. C. for 2 minutes
was performed and then, for the N-terminal region, a cycle of
denaturation at 94.degree. C. for 10 seconds, annealing at
55.degree. C. for 30 seconds and elongation at 68.degree. C. for 30
seconds, and for the C-terminal region, a cycle of denaturation at
94.degree. C. for 10 seconds, annealing at 55.degree. C. for 30
seconds and elongation at 68.degree. C. for 2 minutes, were
repeated 30 times, respectively.
[0149] Next, to obtain a fragment of the pta-ack gene in which an
internal sequence of pta and ack is deleted, the above-mentioned
gene products of the N-terminal region of pta and C-terminal region
of ack were mixed at an approximately equimolar concentration, and
PCR was carried out using the mixture as templates and synthetic
DNAs of SEQ ID NOS: 22 and 18 as primers, to thereby yield
amplified product of a mutation-introduced pta-ack gene. The PCR
was carried out using KOD-plus-(TOYOBO) in such a way that one
cycle of heat-retention at 94.degree. C. for 2 minutes was
performed and then a cycle of denaturation at 94.degree. C. for 10
seconds, annealing at 55.degree. C. for 30 seconds, and elongation
at 68.degree. C. for 2.5 minutes was repeated 30 times, to thereby
yield an amplified product of the mutation-introduced pta-ack gene
of interest. The PCR product thus obtained was purified by a
conventional procedure and then digested with XbaI, followed by
insertion into XbaI site of pBS5T. Competent cells of Escherichia
coli JM109 (Takara Bio Inc.) were used for transformation with this
DNA and transformed cells were applied on an LB medium containing
100 .mu.M of IPTG, 40 .mu.g/ml of X-Gal, and 25 .mu.g/ml of
kanamycin, followed by overnight culture. Subsequently, appeared
white colonies were picked up, and single colonies were then
isolated, thereby transformants were obtained. Plasmids were
extracted from the transformants, and a plasmid into which a PCR
product of interest was inserted was named pBS5T::.DELTA.pta-ack.
FIG. 6 shows the construction procedures of
pBS5T::.DELTA.pta-ack.
(B) Preparation of pta-ack-Disrupted Strain
[0150] The replication origin for coryneform bacteria in
pBSST.:.DELTA.pta-ack obtained by the above-mentioned (A) is
temperature-sensitive. That is, the plasmid is autonomously
replicable in a cell of a coryneform bacterium at 25.degree. C.,
but it is not autonomously replicable at 31.5.degree. C. (or
34.degree. C.). Brevibacterium lactofermentum 2256.DELTA.(ldh)
strain was transformed using the plasmid by the electric pulse
method, and transformed cells were applied on a CM-Dex medium
containing 25 .mu.g/ml of kanamycin, followed by culture at
25.degree. C. for 2 nights. Appeared colonies were isolated, to
thereby yield transformants. The transformants have the plasmid.
The transformants were cultured at 34.degree. C. overnight in a
CM-flex liquid medium not containing kanamycin, and after suitable
dilution, it was applied on a CM-Dex liquid medium containing 25
.mu.g/ml of kanamycin, followed by culture at 34.degree. C. for
about 30 hours. The strain grown on the medium contains the
kanamycin resistance gene and sacB gene which are derived from the
plasmid on the genome, as a result of homologous recombination
between the pta-ack gene fragment on the plasmid and the pta-ack
gene on genome of Brevibacterium lactofermentum 2256.DELTA.(ldh)
strain.
[0151] Next, the single crossover recombinant was cultured at
31.5.degree. C. overnight in CM-flex liquid medium not containing
kanamycin, and after suitable dilution, it was applied on 10%
sucrose-containing Dex-S10 medium not containing kanamycin,
followed by culture at 31.5.degree. C. for about 30 hours. As a
result, about 50 strains, which were considered to become
sucrose-insensitive due to elimination of sacB gene by the second
homologous recombination, were obtained.
[0152] The thus obtained strains include: a strain in which pta and
ack genes were replaced by the mutant type derived from
pBS5T:;.DELTA.pta-ack; and a strain in which pta and ack genes were
reverted to the wild type. Whether the pta and ack genes are the
mutant type or the wild type can be confirmed easily by directly
subjecting a bacterial strain obtained through culture on a Dex-S10
agar medium to PCR and detecting the pta and ack genes. Analysis of
the pta-ack gene by using primers (SEQ ID NOS: 19 and 16) for PCR
amplification should result in a DNA fragment of 5.0 kb for the
wild type and a DNA fragment of 2.7 kb for the mutant type having a
deleted region.
[0153] As a result of the analysis of the sucrose-insensitive
strain by the above-mentioned method, a strain carrying only the
mutant type gene was selected and named 2256.DELTA.(ldh, pta,
ack).
Example 5
Construction of Pyruvate Oxidase Gene-Disrupted Strain
(A) Cloning of A Fragment For Disrupting Pyruvate Oxidase Gene
[0154] A fragment of a pyruvate oxidase gene (hereinafter,
abbreviated as poxB) of Brevibacterium lactofermentum 2256 strain
in which ORF thereof was deleted was obtained by crossover PCR
using as primers synthetic DNAs designed based on the nucleotide
sequence (SEQ ID NO: 42) of the gene of Corynebacterium glutamicum
ATCC13032 (GenBank Database Accession No. NC.sub.--003450), which
has already been disclosed. That is, PCR was carried out using a
genomic DNA of Brevibacterium lactofermentum 2256 strain as a
template and synthetic DNAs of SEQ ID NOS: 23 and 24 as primers,
thereby amplified product of N-terminal region of the poxB gene was
obtained.
[0155] On the other hand, to obtain amplified product of C-terminal
region of the poxB gene, PCR was carried out using a genomic DNA of
Brevibacterium lactofermentum 2256 strain as a template and
synthetic DNAs of SEQ ID NOS: 25 and 26 as primers, SEQ ID NOS: 24
and 25 are complementary to each other. The PCR was carried out
using KOD-plus-(TOYOBO) in such a way that one cycle of
heat-retention at 94.degree. C. for 2 minutes was performed and
then a cycle of denaturation at 94.degree. C. for 10 seconds,
annealing at 55.degree. C. for 30 seconds and elongation at
68.degree. C. for 40 seconds was repeated 30 times for both the
N-terminal region and the C-terminal region Next, to obtain a
fragment of poxB gene in which its internal sequence is deleted,
the above-mentioned gene products of N-terminal and C-terminal
regions of poxB were mixed at an approximate equimolar
concentration, and PCR was carried out using the mixture as
templates and synthetic DNAs of SEQ ID NOS: 27 and 28 as primers,
to thereby yield amplified product of a mutation-introduced poxB
gene. The PCR was carried out using KOD-plus-(TOYOBO) in such a way
that one cycle of heat-retention at 94.degree. C. for 2 minutes was
performed, and then a cycle of denaturation at 94.degree. C. for 10
seconds, annealing at 55.degree. C. for 30 seconds and elongation
at 68.degree. C. for 70 seconds was repeated 30 times, to thereby
yield an amplified product of the mutation-introduced poxB gene of
interest.
[0156] The PCR product thus obtained was purified by a conventional
procedure and then digested with XbaI, followed by insertion into
XbaI site of pBS5T constructed in Example 1 (C) as described above.
Competent cells of Escherichia coli JM109 (Takara Bio Inc.) were
used for transformation with this DNA and transformed cells were
applied on an LB medium containing 100 .mu.M of IPTC; 40 .mu.g/ml
of X-Gal, and 25 .mu.g/ml of kanamycin, followed by overnight
culture. Subsequently, appeared white colonies were picked up, and
single colonies were isolated, thereby transformants were obtained.
Plasmids were extracted from the transformants, and a plasmid in
which a PCR product of interest was inserted was named
pBS5T::.DELTA.poxB. FIG. 7 shows the construction procedures of
pBS5T::.DELTA.poxB.
(B) Preparation of poxB-Disrupted Strain
[0157] The replication origin for coryneform bacteria in
pBS5T::.DELTA.poxB obtained in Example 5 (A) as described above is
temperature-sensitive. That is, the plasmid is autonomously
replicable in a cell of a coryneform bacterium at 25.degree. C.,
but it is not autonomously replicable at 31.5.degree. C. (or
34.degree. C.). Brevibacterium lactofermentum 2256.DELTA.(ldh, pta,
ack) strain was transformed using the plasmid by the electric pulse
method, and the transformed cells were applied on a CM-Dex medium
containing 25 .mu.g/ml of kanamycin, followed by culture at
25.degree. C. for 2 nights. Appeared colonies were isolated, to
thereby yield transformants. The transformants should have the
plasmid.
[0158] The transformants were cultured at 34.degree. C. overnight
in a CM-Dex liquid medium not containing kanamycin, and after
suitable dilution, it was applied on a CM-Dex medium containing 25
.mu.g/ml of kanamycin, followed by culture at 34.degree. C. for
about 30 hours. In a strain grown on the medium, the kanamycin
resistance gene and sacB gene which are derived from the plasmid
are inserted into the genome, as a result of homologous
recombination between the poxB gene fragment on the plasmid and the
poxB gene on the genome of Brevibacterium lactofermentum
2256.DELTA.(ldh, pta, ack) strain. Next, the single crossover
recombinant was cultured at 31.5.degree. C. overnight in CM-Dex
liquid medium not containing kanamycin, and after suitable
dilution, it was applied on 10% sucrose-containing Dex-S10 medium
not containing kanamycin, followed by culture at 31.5.degree. C.
for about 30 hours. As a result, about 50 strains, which were
considered to become sucrose-insensitive due to elimination of sacB
gene by the second homologous recombination, were obtained.
[0159] The obtained strains include: a strain in which poxB gene
was replaced by the mutant type derived from pBS5T::.DELTA.poxB;
and a strain in which poxB gene reverted to the wild type. Whether
the poxB gene is the mutant type or the wild type can be confirmed
easily by directly subjecting a bacterial strain obtained through
culture on a Dex-S10 agar medium to PCR and detecting the poxB
gene. A DNA fragment of 2.4 kb for the wild type and a DNA fragment
of 1.2 kb for the mutant type having the deleted region can be
detected by analysis of the poxB gene with primers (SEQ ID NOS: 23
and 26) for PCR amplification. As a result of the analysis of the
sucrose-insensitive strain by the above-mentioned method, a strain
carrying only the mutant type gene was selected and named
2256.DELTA.(ldh, pta, ack, poxB).
Example 6
Construction of Acetyl-CoA Hydrolase Gene-Disrupted Strain
(A) Cloning of A Fragment For Disrupting Acetyl-CoA Hydrolase
Gene
[0160] A fragment of acetyl-CoA hydrolase gene (hereinafter,
abbreviated as ach) of Brevibacterium lactofermentum 2256 strain in
which ORF thereof was deleted was obtained by crossover PCR using
as primers synthetic DNAs designed based on the nucleotide sequence
(SEQ ID NO: 44) of the gene of Corynebacterium glutamicum ATCC13032
(GenBank Database Accession No. NC.sub.--003450), which has already
been disclosed. That is, PCR was carried out using a genomic DNA of
Brevibacterium lactofermentum 2256 strain as a template and
synthetic DNAs of SEQ ID NOS: 29 and 30 as primers, thereby
amplified product of C-terminal region of the ach gene was
obtained. On the other hand, to obtain amplified product of
N-terminal region of the ach gene, PCR was carried out using a
genomic DNA of Brevibacterium lactofermentum 2256 as a template and
synthetic DNAs of SEQ ID NOS: 31 and 32 as primers. SEQ ID NO: 30
and 31 are complementary to each other. The PCR was carried out
using KOD-plus-(TOYOBO) in such a way that one cycle of
heat-retention at 94.degree. C. for 2 minutes was performed and
then a cycle of denaturation at 94.degree. C. for 10 seconds,
annealing at 55.degree. C. for 30 seconds and elongation at
68.degree. C. for 50 seconds was repeated 30 times for the
N-terminal region and the C-terminal region. Next, to obtain a
fragment of the ach gene in which an internal sequence is deleted,
the above-mentioned gene products of the N-terminal and C-terminal
regions of ach were mixed at an approximately equimolar
concentration, and PCR was carried out using the mixture as
templates and synthetic DNAs of SEQ ID NOS: 33 and 34 as primers,
to thereby yield amplified product of a mutation-introduced ach
gene. The PCR was carried out using KOD-plus-(TOYOBO) in such a way
that one cycle of heat-retention at 94.degree. C. for 2 minutes was
performed and then a cycle of denaturation at 94.degree. C. for 10
seconds, annealing at 55.degree. C. for 30 seconds and elongation
at 68.degree. C. for 90 seconds was repeated 30 times, to thereby
yield an amplified product of the mutation-introduced ach gene of
interest. The PCR product thus obtained was purified by a
conventional procedure and digested with XbaI, followed by
insertion into XbaI site of pBS4S constructed in Example 1 (B) as
described above. Competent cells of Escherichia coli JM109 (Takara
Bio Inc.) were used for transformation with this DNA and
transformed cells were applied on an LB medium containing 100 .mu.M
of IPTC; 40 .mu.g/ml of X-Gal, and 25 .mu.g/ml of kanamycin,
followed by overnight culture. Subsequently, appeared white
colonies were picked up, and single colonies were isolated, thereby
transformants were obtained. Plasmids were extracted from the
transformants, and a plasmid in which a PCR product of interest was
inserted was named pBS4S::.DELTA.ach. FIG. 8 shows the construction
procedures of pBS4S::.DELTA.ach.
(B) Preparation of ach-Disrupted Strain
[0161] The pBS4S::.DELTA.ach obtained in the above-mentioned (A)
does not include a region which enables autonomous replication in a
cell of a coryneform bacterium, so when a coryneform bacterium is
transformed with the plasmid, a strain in which the plasmid is
integrated into a chromosome by homologous recombination appears at
a very low frequency as a transformant. Brevibacterium
lactofermentum 2256.DELTA.(ldh) strain, 2256.DELTA.(ldh, pta, ack)
strain and 2256.DELTA.(ldh, pta, ack, poxB) strain were transformed
by using a high concentration of the plasmid pBS4S::.DELTA.ach by
the electric pulse method, and transformed cells were applied on a
CM-Dex medium containing 25 .mu.g/ml kanamycin, followed by culture
at 31.5.degree. C. for about 30 hours. In the strain grown on the
medium, the kanamycin resistance gene and sacB gene derived from
the plasmid are inserted on the genome as a result of homologous
recombination between the ach gene fragment on the plasmid and the
ach gene on the genome of each of Brevibacterium lactofermentum
2256.DELTA.(ldh) strain, 2256.DELTA.(ldh, pta, ack) strain and
2256.DELTA.(ldh, pta, ack, poxB) strain.
[0162] Next, the single crossover recombinant was cultured at
31.5.degree. C. overnight in CM-Dex liquid medium not containing
kanamycin, and after suitable dilution, it was applied on 10%
sucrose-containing Dex-S10 medium not containing kanamycin,
followed by culture at 31.5.degree. C. for about 30 hours, As a
result, about 50 strains, which were considered to become
sucrose-insensitive due to elimination of sacB gene by the second
homologous recombination, were obtained.
[0163] The thus obtained strains include: a strain in which ach
gene was replaced by the mutant type derived from
pBS4S::.DELTA.ach; and a strain in which ach gene reverted to the
wild type. Whether the ach gene is the mutant type or the wild type
can be confirmed easily by directly subjecting a bacterial strain
obtained through culture in a Dex-S10 agar medium to PCR and
detecting the ach gene. Analysis of the ach gene by using primers
(SEQ ID NOS: 29 and 32) for PCR amplification should result in a
DNA fragment of 2.9 kb for the wild type and a DNA fragment of 1.4
kb for the mutant type having a deleted region. As a result of the
analysis of the sucrose-insensitive strain by the above-mentioned
method, a strain carrying only the mutant type gene was selected
and strains obtained from 2256.DELTA.(ldh), 2256.DELTA.(ldh, pta,
ack), and 2256.DELTA.(ldh, pta, ack, poxB) were named
2256.DELTA.(ldh, ach), 2256.DELTA.(ldh, pta, ack, ach), and
2256.DELTA.(ldh, pta, ack, poxB, ach), respectively.
Example 7
Succinic Acid Production By the ach-Disrupted Strain
(A) Evaluation of Culture of the ach-Deficient Strain
[0164] Brevibacterium lactofermentum 2256.DELTA.(ldh) strain and
2256.DELTA.(ldh, ach) strain were used for culture for producing
succinic acid as follows. Bacterial cells of the 2256.DELTA.(ldh)
strain and 2256.DELTA.(ldh, ach) strain obtained by culturing them
on a CM-Dex plate medium were inoculated into 3 ml of a seed medium
(10 g/L of glucose, 2.5 g/L of (NH.sub.4).sub.2SO.sub.4, 0.5 g/L of
KH.sub.2PO.sub.4, 0.25 g/L of MgSO.sub.4.7H.sub.2O, 2 g/L of urea,
0.01 g/L of FeSO.sub.4.7H.sub.2O, 0.01 g/L of MnSO.sub.4.7H.sub.2O,
50 .mu.g/L of biotin, 100 .mu.g/L of VB1.HCl, 15 mg/L of
protocatechuic acid, 0.02 mg/L of CuSO.sub.4, and 10 mg/L of
CaCl.sub.2, with pH 7.0 (KOH)). Shaking culture was performed in a
test tube at 31.5.degree. C. for about 15 hours under an aerobic
condition.
[0165] After that, 3 ml of a main medium (70 g/L of glucose, 5 g/L
of (NH.sub.4).sub.2SO.sub.4, 2 g/L of KH.sub.2PO.sub.4, 3 g/L of
urea, 0.01 g/L of FeSO.sub.4.7H.sub.2O, 0.01 g/L of
MnSO.sub.4.7H.sub.2O, 200 .mu.g/L of biotin, 200 .mu.g/L of
VB1.HCl, 40 g/L of MOPS, 50 g/L of MgCO.sub.3, with pH 6.8 (NaOH))
was added into the tube. For preventing aeration, the succinic acid
production culture was carried out while the tube was sealed
hermetically with a silicon cap. The culture was performed by
shaking at 31.5.degree. C. for about 24 hours and terminated before
sugar in the medium was exhausted.
[0166] After completion of the culture, the accumulation amounts of
succinic acid and by-product acetic acid in the medium were
analyzed by liquid chromatography after the culture liquid had been
suitably diluted. A column obtained by connecting two pieces of
Shim-pack SCR-102H (Shimadzu) in series was used, and the sample
was eluted at 40.degree. C. by using 5 mM p-toluene sulfonic acid.
The eluent was neutralized by using 20 mM Bis-Tris aqueous solution
containing 5 mM p-toluene sulfonic acid and 100 .mu.M of EDTA.
Succinic acid and acetic acid were each measured by determining the
electric conductivity by means of CDD-10AD (Shimadzu). The obtained
results are shown in Table 1.
[0167] It was found that yield of the 2256.DELTA.(ldh, ach) strain
improved by about 6% as compared to the 2256.DELTA.(ldh) strain as
a parent strain.
TABLE-US-00001 TABLE 1 Production of succinic acid and acetic acid
in ACH-disrupted strain Consumed Yield of Acetic acid OD620 sugar
succinic (/succinic Strains nm(.times.51) (g/L) acid (%) acid, %)
2256.DELTA.ldh 0.366 35.8 57.1 9.2 2256.DELTA.(ldh, ach) 0.353 31.4
63.1 17.4
(B) Evaluation of Culture of the ach, pta And ack-Disrupted
Strain
[0168] Brevibacterium lactofermentum 2256.DELTA.(ldh) strain and
2256.DELTA.(ldh, ach, pta, ack) strain were used for culture for
producing succinic acid as follows. The bacterial cells of the
2256.DELTA.(ldh) strain and 2256.DELTA.(ldh, ach, pta, ack) strain
obtained by culturing them on a CM-Dex plate medium were inoculated
into 3 ml of the above-mentioned seed medium. Shaking culture was
performed in a test tube at 31.5.degree. C. for about 15 hours
under an aerobic condition.
[0169] After that, 3 ml of the above-mentioned main medium was
added into the test tube. For preventing aeration, the succinic
acid production culture was carried out while the tube was sealed
hermetically with a silicon cap. The culture was performed by
shaking at 31.5.degree. C. for about 24 hours and terminated before
sugar in the medium was exhausted.
[0170] After completion of the culture, the accumulation amounts of
succinic acid and by-product acetic acid in the culture medium were
analyzed by liquid chromatography after the culture medium had been
suitably diluted. A column obtained by connecting two pieces of
Shim-pack SCR-102H (Shimadzu) in series was used, and the sample
was eluted at 40.degree. C. by using 5 mM of p-toluene sulfonic
acid. The eluent was neutralized by using 20 mM of Bis-Tris aqueous
solution containing 5 mM of p-toluene sulfonic acid and 100 .mu.M
of EDTA. The succinic acid and by-product acetic acid were each
measured by determining the electric conductivity by means of
CDD-10AD (Shimadzu). The obtained results are shown in Table 2.
[0171] In the case of 2256.DELTA.(ldh, ach, pta, ack) strain, the
succinic acid production level was equal to the parent strain
2256.DELTA.(ldh), but the level of acetic acid with respect to
succinic acid was reduced to about one third of 2256.DELTA.(ldh,
ach) strain and to about half of 2256.DELTA.(ldh) strain, which
revealed that production of acetic acid as a by-product drastically
decreased. This result and the above-mentioned result described in
(A) indicated that eliminating or decreasing either PTA-ACK or ACH
activity is ineffective for reducing acetic acid, but acetic acid
is drastically reduced by eliminating or decreasing all these
activities. Meanwhile, it is easily assumed that eliminating or
decreasing the activity of PTA or ACK together with ACH activity is
effective for reducing acetic acid. As for succinic acid
production, eliminating or decreasing only ACH activity was found
to be effective.
TABLE-US-00002 TABLE 2 Production of succinic acid and acetic acid
in the strain in which ACH, PTA and ACK have been disrupted in
combination Consumed Yield of Acetic acid OD620 sugar succinic
(/succinic Strains (.times.51) (g/L) acid (%) acid %)
2256.DELTA.ldh 0.342 31.8 57.3 11.9 2256.DELTA.(ldh, ach) 0.364
33.0 63.6 17.3 2256.DELTA.(ldh, ach, 0.347 39.0 58.3 6.1 pta,
ack)
(C) Evaluation of Culture of the Ach, Pta, Ack and PoxB-Disrupted
Strain
[0172] Brevibacterium lactofermentum 2256.DELTA.(ldh) strain,
2256.DELTA.(ldh, pta-ack, ach) strain and 2256.DELTA.(ldh, pta-ack,
poxB, ach) strain were used for culture for producing succinic acid
as follows. The bacterial cells of the 2256.DELTA.(ldh),
2256.DELTA.(ldh, pta-ack, ach) strain and 2256.DELTA.(ldh, pta-ack,
poxB, ach) strain obtained by culturing them on a CM-Dex plate
medium were inoculated into 3 ml of the above-mentioned seed
medium, and shaking culture in a test tube was performed at
31.5.degree. C. for about 15 hours under an aerobic condition.
[0173] After that, 3 ml of the above-mentioned main medium was
added into the tube. For preventing aeration, the succinic acid
production culture was carried out while the tube was sealed
hermetically with a silicon cap. The culture was performed by
shaking at 31.5.degree. C. for about 24 hours and terminated before
sugar in the medium was exhausted. After completion of the culture,
the accumulation amounts of succinic acid and by-product acetic
acid in the medium were analyzed by liquid chromatography after the
medium had been suitably diluted. A column obtained by connecting
two pieces of Shim-pack SCR-102H (Shimadzu) in series, and the
sample was eluted at 40.degree. C. by using 5 mM of p-toluene
sulfonic acid. The eluent was neutralized by using 20 mM of
Bis-Tris aqueous solution containing 5 mM of p-toluene sulfonic
acid and 100 .mu.M of EDTA. The succinic acid and by-product acetic
acid were each measured by determining the electric conductivity by
means of CDD-10AD (Shimadzu), The obtained results are shown in
Table 3.
[0174] In the case of 2256.DELTA.(ldh, pta, ack, ach, poxB) strain
prepared by further disrupting poxB in the 2256.DELTA.(ldh, pta,
ack, ach) strain, acetic acid was further decreased by about 40% as
compared to the parent 2256.DELTA.(ldh, pta, ack, ach) strain. The
result revealed that eliminating or decreasing ach activity
together with activities of all or any of pta, ack and poxB is
effective for reducing acetic acid.
TABLE-US-00003 TABLE 3 Production of succinic acid and acetic acid
in the strain in which ACH, PTA, ACK and POXB have been decreased
in combination Consumed Yield of Acetic acid OD620 sugar succinic
(/succinic Strains (.times.51) (g/L) acid (%) acid %)
2256.DELTA.ldh 0.342 31.8 57.3 11.9 2256.DELTA.(ldh, pta, 0.347 39
58.4 6.1 ack, ach) 2256.DELTA.(ldh, pta, 0.372 39.8 56.1 3.6 ack,
poxB, ach)
INDUSTRIAL APPLICABILITY
[0175] Use of the bacterium of the present invention enables
efficient production of succinic acid. Succinic acid is useful as a
raw material for a biodegradable polymer and the like.
[0176] While the invention has been described in detail with
reference to preferred embodiments thereof, it will be apparent to
one skilled in the art that various changes can be made, and
equivalents employed, without departing from the scope of the
invention. Each of the aforementioned documents, including the
foreign priority document, JP 2004-150658, is incorporated by
reference herein in its entirety.
Sequence CWU 1
1
47124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1cgggatcctt tttaacccat caca 24229DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2gaagatcttc aaaaggttag gaatacggt 29323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3ccttttgaag atcgaccagt tgg 23444DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 4tacctggaat gctgttttcc
cagggatcgc agtggtgagt aacc 44528DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 5cctgggaaaa cagcattcca
ggtattag 28623DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 6tgcaggtcga ctctagagga tcc
23720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7cactgcacgg ccctgcgaac 20842DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8cgccaactag gcgccaaaaa ttcctgattt ccctaaccgg ac 42942DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9gtccggttag ggaaatcagg aatttttggc gcctagttgg cg 421020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10tgtgggcctt cggcgaggac 201126DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 11gagtcgaccg caccccattt ttcata
261228DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 12tggtcgacgt gaatgctcgg cgggatcc
281324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13cttccatctt cctcatggtg ctgc 241444DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14ccaggagagc taagcgaact ccattagctg cgtcctcctg cctg
441544DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15caggcaggag gacgcagcta atggagttcg cttagctctc ctgg
441631DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16gcgtctagac ctttaggagt gcgatgtccc c
311733DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 17gcgtctagac gactgtgctg ttaacccgaa ccc
331833DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 18gcgtctagag agttaggccc ttagaagcga ttc
331924DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 19gctcaaagcg tggaattgag atcg 242042DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20ccaggagagc taagcgaact ttcggcgctc atgactggtt cg
422142DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 21cgaaccagtc atgagcgccg aaagttcgct tagctctcct gg
422230DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 22gcgtctagag tacgcaaggc ggacgaacgc
302323DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 23gccttgatat cttcccgcaa acc 232447DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
24cttgtggtcc tggaaacaca caccgaagtg aatttcgcag agattgc
472548DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25cgcaatctct gcgaaattca cttcggtgtg tgtttccagg
accacaag 482622DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 26ggtttctcgg ggtctaaacc gg
222733DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 27gggaatctag accacgccaa tggaaatttc tcc
332835DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28gggaatctag acgtgacaag atctggcgaa atcgc
352924DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 29gcttctgcgc aaagcaagcc tccg 243050DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
30gtccgattac ctgaggaggt attcccatga aggcataagt tttttcttgg
503150DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 31ccaagaaaaa acttatgcct tcatgggaat acctcctcag
gtaatcggac 503226DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 32ggtcatgtgc atggttttct cattgc
263333DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 33ggcctctaga cctgcaccga tcaggatgag tgg
333435DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 34gcgctctaga ctcaacaaga gcacgcgcag tcacc
35352014DNABacillus subtilisCDS(464)..(1882) 35gatccttttt
aacccatcac atatacctgc cgttcactat tatttagtga aatgagatat 60tatgatattt
tctgaattgt gattaaaaag gcaactttat gcccatgcaa cagaaactat
120aaaaaataca gagaatgaaa agaaacagat agatttttta gttctttagg
cccgtagtct 180gcaaatcctt ttatgatttt ctatcaaaca aaagaggaaa
atagaccagt tgcaatccaa 240acgagagtct aatagaatga ggtcgaaaag
taaatcgcgc gggtttgtta ctgataaagc 300aggcaagacc taaaatgtgt
aaagggcaaa gtgtatactt tggcgtcacc ccttacatat 360tttaggtctt
tttttattgt gcgtaactaa cttgccatct tcaaacagga gggctggaag
420aagcagaccg ctaacacagt acataaaaaa ggagacatga acg atg aac atc aaa
475 Met Asn Ile Lys 1aag ttt gca aaa caa gca aca gta tta acc ttt
act acc gca ctg ctg 523Lys Phe Ala Lys Gln Ala Thr Val Leu Thr Phe
Thr Thr Ala Leu Leu5 10 15 20gca gga ggc gca act caa gcg ttt gcg
aaa gaa acg aac caa aag cca 571Ala Gly Gly Ala Thr Gln Ala Phe Ala
Lys Glu Thr Asn Gln Lys Pro 25 30 35tat aag gaa aca tac ggc att tcc
cat att aca cgc cat gat atg ctg 619Tyr Lys Glu Thr Tyr Gly Ile Ser
His Ile Thr Arg His Asp Met Leu 40 45 50caa atc cct gaa cag caa aaa
aat gaa aaa tat caa gtt cct gaa ttc 667Gln Ile Pro Glu Gln Gln Lys
Asn Glu Lys Tyr Gln Val Pro Glu Phe 55 60 65gat tcg tcc aca att aaa
aat atc tct tct gca aaa ggc ctg gac gtt 715Asp Ser Ser Thr Ile Lys
Asn Ile Ser Ser Ala Lys Gly Leu Asp Val 70 75 80tgg gac agc tgg cca
tta caa aac gct gac ggc act gtc gca aac tat 763Trp Asp Ser Trp Pro
Leu Gln Asn Ala Asp Gly Thr Val Ala Asn Tyr85 90 95 100cac ggc tac
cac atc gtc ttt gca tta gcc gga gat cct aaa aat gcg 811His Gly Tyr
His Ile Val Phe Ala Leu Ala Gly Asp Pro Lys Asn Ala 105 110 115gat
gac aca tcg att tac atg ttc tat caa aaa gtc ggc gaa act tct 859Asp
Asp Thr Ser Ile Tyr Met Phe Tyr Gln Lys Val Gly Glu Thr Ser 120 125
130att gac agc tgg aaa aac gct ggc cgc gtc ttt aaa gac agc gac aaa
907Ile Asp Ser Trp Lys Asn Ala Gly Arg Val Phe Lys Asp Ser Asp Lys
135 140 145ttc gat gca aat gat tct atc cta aaa gac caa aca caa gaa
tgg tca 955Phe Asp Ala Asn Asp Ser Ile Leu Lys Asp Gln Thr Gln Glu
Trp Ser 150 155 160ggt tca gcc aca ttt aca tct gac gga aaa atc cgt
tta ttc tac act 1003Gly Ser Ala Thr Phe Thr Ser Asp Gly Lys Ile Arg
Leu Phe Tyr Thr165 170 175 180gat ttc tcc ggt aaa cat tac ggc aaa
caa aca ctg aca act gca caa 1051Asp Phe Ser Gly Lys His Tyr Gly Lys
Gln Thr Leu Thr Thr Ala Gln 185 190 195gtt aac gta tca gca tca gac
agc tct ttg aac atc aac ggt gta gag 1099Val Asn Val Ser Ala Ser Asp
Ser Ser Leu Asn Ile Asn Gly Val Glu 200 205 210gat tat aaa tca atc
ttt gac ggt gac gga aaa acg tat caa aat gta 1147Asp Tyr Lys Ser Ile
Phe Asp Gly Asp Gly Lys Thr Tyr Gln Asn Val 215 220 225cag cag ttc
atc gat gaa ggc aac tac agc tca ggc gac aac cat acg 1195Gln Gln Phe
Ile Asp Glu Gly Asn Tyr Ser Ser Gly Asp Asn His Thr 230 235 240ctg
aga gat cct cac tac gta gaa gat aaa ggc cac aaa tac tta gta 1243Leu
Arg Asp Pro His Tyr Val Glu Asp Lys Gly His Lys Tyr Leu Val245 250
255 260ttt gaa gca aac act gga act gaa gat ggc tac caa ggc gaa gaa
tct 1291Phe Glu Ala Asn Thr Gly Thr Glu Asp Gly Tyr Gln Gly Glu Glu
Ser 265 270 275tta ttt aac aaa gca tac tat ggc aaa agc aca tca ttc
ttc cgt caa 1339Leu Phe Asn Lys Ala Tyr Tyr Gly Lys Ser Thr Ser Phe
Phe Arg Gln 280 285 290gaa agt caa aaa ctt ctg caa agc gat aaa aaa
cgc acg gct gag tta 1387Glu Ser Gln Lys Leu Leu Gln Ser Asp Lys Lys
Arg Thr Ala Glu Leu 295 300 305gca aac ggc gct ctc ggt atg att gag
cta aac gat gat tac aca ctg 1435Ala Asn Gly Ala Leu Gly Met Ile Glu
Leu Asn Asp Asp Tyr Thr Leu 310 315 320aaa aaa gtg atg aaa ccg ctg
att gca tct aac aca gta aca gat gaa 1483Lys Lys Val Met Lys Pro Leu
Ile Ala Ser Asn Thr Val Thr Asp Glu325 330 335 340att gaa cgc gcg
aac gtc ttt aaa atg aac ggc aaa tgg tac ctg ttc 1531Ile Glu Arg Ala
Asn Val Phe Lys Met Asn Gly Lys Trp Tyr Leu Phe 345 350 355act gac
tcc cgc gga tca aaa atg acg att gac ggc att acg tct aac 1579Thr Asp
Ser Arg Gly Ser Lys Met Thr Ile Asp Gly Ile Thr Ser Asn 360 365
370gat att tac atg ctt ggt tat gtt tct aat tct tta act ggc cca tac
1627Asp Ile Tyr Met Leu Gly Tyr Val Ser Asn Ser Leu Thr Gly Pro Tyr
375 380 385aag ccg ctg aac aaa act ggc ctt gtg tta aaa atg gat ctt
gat cct 1675Lys Pro Leu Asn Lys Thr Gly Leu Val Leu Lys Met Asp Leu
Asp Pro 390 395 400aac gat gta acc ttt act tac tca cac ttc gct gta
cct caa gcg aaa 1723Asn Asp Val Thr Phe Thr Tyr Ser His Phe Ala Val
Pro Gln Ala Lys405 410 415 420gga aac aat gtc gtg att aca agc tat
atg aca aac aga gga ttc tac 1771Gly Asn Asn Val Val Ile Thr Ser Tyr
Met Thr Asn Arg Gly Phe Tyr 425 430 435gca gac aaa caa tca acg ttt
gcg cca agc ttc ctg ctg aac atc aaa 1819Ala Asp Lys Gln Ser Thr Phe
Ala Pro Ser Phe Leu Leu Asn Ile Lys 440 445 450ggc aag aaa aca tct
gtt gtc aaa gac agc atc ctt gaa caa gga caa 1867Gly Lys Lys Thr Ser
Val Val Lys Asp Ser Ile Leu Glu Gln Gly Gln 455 460 465tta aca gtt
aac aaa taaaaacgca aaagaaaatg ccgatatcct attggcattt 1922Leu Thr Val
Asn Lys 470tcttttattt cttatcaaca taaaggtgaa tcccatatga actatataaa
agcaggcaaa 1982tggctaaccg tattcctaac cttttgaaga tc
201436473PRTBacillus subtilis 36Met Asn Ile Lys Lys Phe Ala Lys Gln
Ala Thr Val Leu Thr Phe Thr1 5 10 15Thr Ala Leu Leu Ala Gly Gly Ala
Thr Gln Ala Phe Ala Lys Glu Thr 20 25 30Asn Gln Lys Pro Tyr Lys Glu
Thr Tyr Gly Ile Ser His Ile Thr Arg 35 40 45His Asp Met Leu Gln Ile
Pro Glu Gln Gln Lys Asn Glu Lys Tyr Gln 50 55 60Val Pro Glu Phe Asp
Ser Ser Thr Ile Lys Asn Ile Ser Ser Ala Lys65 70 75 80Gly Leu Asp
Val Trp Asp Ser Trp Pro Leu Gln Asn Ala Asp Gly Thr 85 90 95Val Ala
Asn Tyr His Gly Tyr His Ile Val Phe Ala Leu Ala Gly Asp 100 105
110Pro Lys Asn Ala Asp Asp Thr Ser Ile Tyr Met Phe Tyr Gln Lys Val
115 120 125Gly Glu Thr Ser Ile Asp Ser Trp Lys Asn Ala Gly Arg Val
Phe Lys 130 135 140Asp Ser Asp Lys Phe Asp Ala Asn Asp Ser Ile Leu
Lys Asp Gln Thr145 150 155 160Gln Glu Trp Ser Gly Ser Ala Thr Phe
Thr Ser Asp Gly Lys Ile Arg 165 170 175Leu Phe Tyr Thr Asp Phe Ser
Gly Lys His Tyr Gly Lys Gln Thr Leu 180 185 190Thr Thr Ala Gln Val
Asn Val Ser Ala Ser Asp Ser Ser Leu Asn Ile 195 200 205Asn Gly Val
Glu Asp Tyr Lys Ser Ile Phe Asp Gly Asp Gly Lys Thr 210 215 220Tyr
Gln Asn Val Gln Gln Phe Ile Asp Glu Gly Asn Tyr Ser Ser Gly225 230
235 240Asp Asn His Thr Leu Arg Asp Pro His Tyr Val Glu Asp Lys Gly
His 245 250 255Lys Tyr Leu Val Phe Glu Ala Asn Thr Gly Thr Glu Asp
Gly Tyr Gln 260 265 270Gly Glu Glu Ser Leu Phe Asn Lys Ala Tyr Tyr
Gly Lys Ser Thr Ser 275 280 285Phe Phe Arg Gln Glu Ser Gln Lys Leu
Leu Gln Ser Asp Lys Lys Arg 290 295 300Thr Ala Glu Leu Ala Asn Gly
Ala Leu Gly Met Ile Glu Leu Asn Asp305 310 315 320Asp Tyr Thr Leu
Lys Lys Val Met Lys Pro Leu Ile Ala Ser Asn Thr 325 330 335Val Thr
Asp Glu Ile Glu Arg Ala Asn Val Phe Lys Met Asn Gly Lys 340 345
350Trp Tyr Leu Phe Thr Asp Ser Arg Gly Ser Lys Met Thr Ile Asp Gly
355 360 365Ile Thr Ser Asn Asp Ile Tyr Met Leu Gly Tyr Val Ser Asn
Ser Leu 370 375 380Thr Gly Pro Tyr Lys Pro Leu Asn Lys Thr Gly Leu
Val Leu Lys Met385 390 395 400Asp Leu Asp Pro Asn Asp Val Thr Phe
Thr Tyr Ser His Phe Ala Val 405 410 415Pro Gln Ala Lys Gly Asn Asn
Val Val Ile Thr Ser Tyr Met Thr Asn 420 425 430Arg Gly Phe Tyr Ala
Asp Lys Gln Ser Thr Phe Ala Pro Ser Phe Leu 435 440 445Leu Asn Ile
Lys Gly Lys Lys Thr Ser Val Val Lys Asp Ser Ile Leu 450 455 460Glu
Gln Gly Gln Leu Thr Val Asn Lys465 470372820DNACorynebacterium
glutamicumCDS(898)..(1851) 37tgcagaatta tgcaagatgc gccgcaacaa
aacgcgatcg gccaaggtca aagtggtcaa 60tgtaatgacc gaaaccgctg cgatgaaact
tatccacggc ggtaaaaacc tctcaattag 120gagcttgacc tcattaatac
tgtgctgggt taattcgccg gtgatcagca gcgcgccgta 180ccccaaggtg
ccgacactaa tgcccgcgat cgtctccttc ggtccaaaat tcttctgccc
240aatcagccgg atttgggtgc gatgcctgat caatcccaca accgtggtgg
tcaacgtgat 300ggcaccagtt gcgatgtggg tggcgttgta aattttcctg
gatacccgcc ggttggttct 360ggggaggatc gagtggattc ccgtcgctgc
cgcatgcccc accgcttgta aaacagccag 420gttagcagcc gtaacccacc
acggtttcgg caacaatgac ggcgagagag cccaccacat 480tgcgatttcc
gctccgataa agccagcgcc catatttgca gggaggattc gcctgcggtt
540tggcgacatt cggatccccg gaactagctc tgcaatgacc tgcgcgccga
gggaggcgag 600gtgggtggca ggttttagtg cgggtttaag cgttgccagg
cgagtggtga gcagagacgc 660tagtctgggg agcgaaacca tattgagtca
tcttggcaga gcatgcacaa ttctgcaggg 720cataggttgg ttttgctcga
tttacaatgt gattttttca acaaaaataa cacttggtct 780gaccacattt
tcggacataa tcgggcataa ttaaaggtgt aacaaaggaa tccgggcaca
840agctcttgct gattttctga gctgctttgt gggttgtccg gttagggaaa tcaggaa
897gtg gga tcg aaa atg aaa gaa acc gtc ggt aac aag att gtc ctc att
945Val Gly Ser Lys Met Lys Glu Thr Val Gly Asn Lys Ile Val Leu Ile1
5 10 15ggc gca gga gat gtt gga gtt gca tac gca tac gca ctg atc aac
cag 993Gly Ala Gly Asp Val Gly Val Ala Tyr Ala Tyr Ala Leu Ile Asn
Gln 20 25 30ggc atg gca gat cac ctt gcg atc atc gac atc gat gaa aag
aaa ctc 1041Gly Met Ala Asp His Leu Ala Ile Ile Asp Ile Asp Glu Lys
Lys Leu 35 40 45gaa ggc aac gtc atg gac tta aac cat ggt gtt gtg tgg
gcc gat tcc 1089Glu Gly Asn Val Met Asp Leu Asn His Gly Val Val Trp
Ala Asp Ser 50 55 60cgc acc cgc gtc acc aag ggc acc tac gct gac tgc
gaa gac gca gcc 1137Arg Thr Arg Val Thr Lys Gly Thr Tyr Ala Asp Cys
Glu Asp Ala Ala65 70 75 80atg gtt gtc att tgt gcc ggc gca gcc caa
aag cca ggc gag acc cgc 1185Met Val Val Ile Cys Ala Gly Ala Ala Gln
Lys Pro Gly Glu Thr Arg 85 90 95ctc cag ctg gtg gac aaa aac gtc aag
att atg aaa tcc atc gtc ggc 1233Leu Gln Leu Val Asp Lys Asn Val Lys
Ile Met Lys Ser Ile Val Gly 100 105 110gat gtc atg gac agc gga ttc
gac ggc atc ttc ctc gtg gcg tcc aac 1281Asp Val Met Asp Ser Gly Phe
Asp Gly Ile Phe Leu Val Ala Ser Asn 115 120 125cca gtg gat atc ctg
acc tac gca gtg tgg aaa ttc tcc ggc ttg gaa 1329Pro Val Asp Ile Leu
Thr Tyr Ala Val Trp Lys Phe Ser Gly Leu Glu 130
135 140tgg aac cgc gtg atc ggc tcc gga act gtc ctg gac tcc gct cga
ttc 1377Trp Asn Arg Val Ile Gly Ser Gly Thr Val Leu Asp Ser Ala Arg
Phe145 150 155 160cgc tac atg ctg ggc gaa ctc tac gaa gtg gca cca
agc tcc gtc cac 1425Arg Tyr Met Leu Gly Glu Leu Tyr Glu Val Ala Pro
Ser Ser Val His 165 170 175gcc tac atc atc ggc gaa cac ggc gac act
gaa ctt cca gtc ctg tcc 1473Ala Tyr Ile Ile Gly Glu His Gly Asp Thr
Glu Leu Pro Val Leu Ser 180 185 190tcc gcg acc atc gca ggc gta tcg
ctt agc cga atg ctg gac aaa gac 1521Ser Ala Thr Ile Ala Gly Val Ser
Leu Ser Arg Met Leu Asp Lys Asp 195 200 205cca gag ctt gag ggc cgt
cta gag aaa att ttc gaa gac acc cgc gac 1569Pro Glu Leu Glu Gly Arg
Leu Glu Lys Ile Phe Glu Asp Thr Arg Asp 210 215 220gct gcc tat cac
att atc gac gcc aag ggc tcc act tcc tac ggc atc 1617Ala Ala Tyr His
Ile Ile Asp Ala Lys Gly Ser Thr Ser Tyr Gly Ile225 230 235 240ggc
atg ggt ctt gct cgc atc acc cgc gca atc ctg cag aac caa gac 1665Gly
Met Gly Leu Ala Arg Ile Thr Arg Ala Ile Leu Gln Asn Gln Asp 245 250
255gtt gca gtc cca gtc tct gca ctg ctc cac ggt gaa tac ggt gag gaa
1713Val Ala Val Pro Val Ser Ala Leu Leu His Gly Glu Tyr Gly Glu Glu
260 265 270gac atc tac atc ggc acc cca gct gtg gtg aac cgc cga ggc
atc cgc 1761Asp Ile Tyr Ile Gly Thr Pro Ala Val Val Asn Arg Arg Gly
Ile Arg 275 280 285cgc gtt gtc gaa cta gaa atc acc gac cac gag atg
gaa cgc ttc aag 1809Arg Val Val Glu Leu Glu Ile Thr Asp His Glu Met
Glu Arg Phe Lys 290 295 300cat tcc gca aat acc ctg cgc gaa att cag
aag cag ttc ttc 1851His Ser Ala Asn Thr Leu Arg Glu Ile Gln Lys Gln
Phe Phe305 310 315taaatctttg gcgcctagtt ggcgacgcaa gtgtttcatt
ggaacacttg cgctgccaac 1911tttttggttt acgggcacaa tgaaactgtt
ggatggaatt tagagtgttt gtagcttaag 1971gagctcaaat gaatgagttt
gaccaggaca ttctccagga gatcaagact gaactcgacg 2031agttaattct
agaacttgat gaggtgacac aaactcacag cgaggccatc gggcaggtct
2091ccccaaccca ttacgttggt gcccgcaacc tcatgcatta cgcgcatctt
cgcaccaaag 2151acctccgtgg cctgcagcaa cgcctctcct ctgtgggagc
tacccgcttg actaccaccg 2211aaccagcagt gcaggcccgc ctcaaggccg
cccgcaatgt tatcggagct ttcgcaggtg 2271aaggcccact ttatccaccc
tcagatgtcg tcgatgcctt cgaagatgcc gatgagattc 2331tcgacgagca
cgccgaaatt ctccttggcg aacccctacc ggatactcca tcctgcatca
2391tggtcaccct gcccaccgaa gccgccaccg acattgaact tgtccgtggc
ttcgccaaaa 2451gcggcatgaa tctagctcgc atcaactgtg cacacgacga
tgaaaccgtc tggaagcaga 2511tgatcgacaa cgtccacacc gttgcagaag
aagttggccg ggaaatccgc gtcagcatgg 2571acctcgccgg accaaaagta
cgcaccggcg aaatcgcccc aggcgcagaa gtaggtcgcg 2631cacgagtaac
ccgcgacgaa accggaaaag tactgacgcc cgcaaaactg tggatcaccg
2691cccacggctc cgaaccagtc ccagcccccg aaagcctgcc cggtcgcccc
gctctgccga 2751ttgaagtcac cccagaatgg ttcgacaaac tagaaatcgg
cagcgtcatc aacgtcccag 2811acacccgcg 282038318PRTCorynebacterium
glutamicum 38Val Gly Ser Lys Met Lys Glu Thr Val Gly Asn Lys Ile
Val Leu Ile1 5 10 15Gly Ala Gly Asp Val Gly Val Ala Tyr Ala Tyr Ala
Leu Ile Asn Gln 20 25 30Gly Met Ala Asp His Leu Ala Ile Ile Asp Ile
Asp Glu Lys Lys Leu 35 40 45Glu Gly Asn Val Met Asp Leu Asn His Gly
Val Val Trp Ala Asp Ser 50 55 60Arg Thr Arg Val Thr Lys Gly Thr Tyr
Ala Asp Cys Glu Asp Ala Ala65 70 75 80Met Val Val Ile Cys Ala Gly
Ala Ala Gln Lys Pro Gly Glu Thr Arg 85 90 95Leu Gln Leu Val Asp Lys
Asn Val Lys Ile Met Lys Ser Ile Val Gly 100 105 110Asp Val Met Asp
Ser Gly Phe Asp Gly Ile Phe Leu Val Ala Ser Asn 115 120 125Pro Val
Asp Ile Leu Thr Tyr Ala Val Trp Lys Phe Ser Gly Leu Glu 130 135
140Trp Asn Arg Val Ile Gly Ser Gly Thr Val Leu Asp Ser Ala Arg
Phe145 150 155 160Arg Tyr Met Leu Gly Glu Leu Tyr Glu Val Ala Pro
Ser Ser Val His 165 170 175Ala Tyr Ile Ile Gly Glu His Gly Asp Thr
Glu Leu Pro Val Leu Ser 180 185 190Ser Ala Thr Ile Ala Gly Val Ser
Leu Ser Arg Met Leu Asp Lys Asp 195 200 205Pro Glu Leu Glu Gly Arg
Leu Glu Lys Ile Phe Glu Asp Thr Arg Asp 210 215 220Ala Ala Tyr His
Ile Ile Asp Ala Lys Gly Ser Thr Ser Tyr Gly Ile225 230 235 240Gly
Met Gly Leu Ala Arg Ile Thr Arg Ala Ile Leu Gln Asn Gln Asp 245 250
255Val Ala Val Pro Val Ser Ala Leu Leu His Gly Glu Tyr Gly Glu Glu
260 265 270Asp Ile Tyr Ile Gly Thr Pro Ala Val Val Asn Arg Arg Gly
Ile Arg 275 280 285Arg Val Val Glu Leu Glu Ile Thr Asp His Glu Met
Glu Arg Phe Lys 290 295 300His Ser Ala Asn Thr Leu Arg Glu Ile Gln
Lys Gln Phe Phe305 310 315394200DNACorynebacterium
glutamicumCDS(956)..(1942)CDS(1945)..(3135) 39cctgctggac ctgcaccgac
aacggcaaca cgcaaagggc gagacatata aagttcgatt 60ccttaaaggg gttctaaaaa
atgtggagta tgtgagcggg ggttccactt gtagattcga 120ctcctatcgg
ggtgcgactg ctaatggtgc cctgctatca accctccatg atacgtggta
180agtgcagact aataaaggcc agtcggggag tattgggggc tttgctgggg
tcagatttgt 240cacgctgcgc gctttcatag accccattaa tggggggtga
agagctgtaa agtaccgcta 300aaaactttgc aaagggtgct tcgcaacttg
taaccgctcc gtattgtttt ctacggcaat 360aagcatttgt gctgctcaaa
gcgtggaatt gagatcggtt tgaaaattac aaaataaaac 420tttgcaaacc
gggctgtacg caaggcggac gaacgctaaa ctatgtaaga aatcacaacc
480tcccctcatt agtgccagga ggcacaagcc tgaagtgtca tcaatgagaa
ggttcaggct 540gaaattagaa aggcgatgta tgtctgacac accgacctca
gctctgatca ccacggtcaa 600ccgcagcttc gatggattcg atttggaaga
agtagcagca gaccttggag ttcggctcac 660ctacctgccc gacgaagaac
tagaagtatc caaagttctc gcggcggacc tcctcgctga 720ggggccagct
ctcatcatcg gtgtaggaaa cacgtttttc gacgcccagg tcgccgctgc
780cctcggcgtc ccagtgctac tgctggtaga caagcaaggc aagcacgttg
ctcttgctcg 840cacccaggta aacaatgccg gcgcagttgt tgcagcagca
tttaccgctg aacaagagcc 900aatgccggat aagctgcgca aggctgtgcg
caaccacagc aacctcgaac cagtc atg 958 Met 1agc gcc gaa ctc ttt gaa
aac tgg ctg ctc aag cgc gca cgc gca gag 1006Ser Ala Glu Leu Phe Glu
Asn Trp Leu Leu Lys Arg Ala Arg Ala Glu 5 10 15cac tcc cac att gtg
ctg cca gaa ggt gac gac gac cgc atc ttg atg 1054His Ser His Ile Val
Leu Pro Glu Gly Asp Asp Asp Arg Ile Leu Met 20 25 30gct gcc cac cag
ctg ctt gat caa gac atc tgt gac atc acg atc ctg 1102Ala Ala His Gln
Leu Leu Asp Gln Asp Ile Cys Asp Ile Thr Ile Leu 35 40 45ggc gat cca
gta aag atc aag gag cgc gct acc gaa ctt ggc ctg cac 1150Gly Asp Pro
Val Lys Ile Lys Glu Arg Ala Thr Glu Leu Gly Leu His50 55 60 65ctt
aac act gca tac ctg gtc aat ccg ctg aca gat cct cgc ctg gag 1198Leu
Asn Thr Ala Tyr Leu Val Asn Pro Leu Thr Asp Pro Arg Leu Glu 70 75
80gaa ttc gcc gaa caa ttc gcg gag ctg cgc aag tca aag agc gtc act
1246Glu Phe Ala Glu Gln Phe Ala Glu Leu Arg Lys Ser Lys Ser Val Thr
85 90 95atc gat gaa gcc cgc gaa atc atg aag gat att tcc tac ttc ggc
acc 1294Ile Asp Glu Ala Arg Glu Ile Met Lys Asp Ile Ser Tyr Phe Gly
Thr 100 105 110atg atg gtc cac aac ggc gac gcc gac gga atg gta tcc
ggt gca gca 1342Met Met Val His Asn Gly Asp Ala Asp Gly Met Val Ser
Gly Ala Ala 115 120 125aac acc acc gca cac acc att aag cca agc ttc
cag atc atc aaa act 1390Asn Thr Thr Ala His Thr Ile Lys Pro Ser Phe
Gln Ile Ile Lys Thr130 135 140 145gtt cca gaa gca tcc gtc gtt tct
tcc atc ttc ctc atg gtg ctg cgc 1438Val Pro Glu Ala Ser Val Val Ser
Ser Ile Phe Leu Met Val Leu Arg 150 155 160ggg cga ctg tgg gca ttc
ggc gac tgt gct gtt aac ccg aac cca act 1486Gly Arg Leu Trp Ala Phe
Gly Asp Cys Ala Val Asn Pro Asn Pro Thr 165 170 175gct gaa cag ctt
ggt gaa atc gcc gtt gtg tca gca aaa act gca gca 1534Ala Glu Gln Leu
Gly Glu Ile Ala Val Val Ser Ala Lys Thr Ala Ala 180 185 190caa ttt
ggc att gat cct cgc gta gcc atc ttg tcc tac tcc act ggc 1582Gln Phe
Gly Ile Asp Pro Arg Val Ala Ile Leu Ser Tyr Ser Thr Gly 195 200
205aac tcc ggc gga ggc tca gat gtg gat cgc gcc atc gac gct ctt gca
1630Asn Ser Gly Gly Gly Ser Asp Val Asp Arg Ala Ile Asp Ala Leu
Ala210 215 220 225gaa gca cgc cga ctt aac cca gaa cta tgc gtc gat
gga cca ctt cag 1678Glu Ala Arg Arg Leu Asn Pro Glu Leu Cys Val Asp
Gly Pro Leu Gln 230 235 240ttc gac gcc gcc gtc gac ccg ggt gtg gcg
cgc aag aag atg cca gac 1726Phe Asp Ala Ala Val Asp Pro Gly Val Ala
Arg Lys Lys Met Pro Asp 245 250 255tct gac gtc gct ggc cag gca aat
gtg ttt atc ttc cct gac ctg gaa 1774Ser Asp Val Ala Gly Gln Ala Asn
Val Phe Ile Phe Pro Asp Leu Glu 260 265 270gcc gga aac atc ggc tac
aaa act gca caa cgc acc ggt cac gcc ctg 1822Ala Gly Asn Ile Gly Tyr
Lys Thr Ala Gln Arg Thr Gly His Ala Leu 275 280 285gca gtt ggt ccg
att ctg cag ggc cta aac aaa cca gtc aac gac ctt 1870Ala Val Gly Pro
Ile Leu Gln Gly Leu Asn Lys Pro Val Asn Asp Leu290 295 300 305tcc
cgt ggc gca aca gtc cct gac atc gtc aac aca gta gcc atc aca 1918Ser
Arg Gly Ala Thr Val Pro Asp Ile Val Asn Thr Val Ala Ile Thr 310 315
320gca att cag gca gga gga cgc agc ta atg gca ttg gca ctt gtt ttg
1965Ala Ile Gln Ala Gly Gly Arg Ser Met Ala Leu Ala Leu Val Leu 325
330 335aac tcc ggt tca tct tcc atc aaa ttc cag ctg gtc aac ccc gaa
aac 2013Asn Ser Gly Ser Ser Ser Ile Lys Phe Gln Leu Val Asn Pro Glu
Asn 340 345 350tct gcc atc gac gag cca tat gtt tct ggt ctt gtg gag
cag att ggt 2061Ser Ala Ile Asp Glu Pro Tyr Val Ser Gly Leu Val Glu
Gln Ile Gly 355 360 365gag cca aac ggc cgc atc gta ctc aaa ata gag
ggt gaa aaa tat acc 2109Glu Pro Asn Gly Arg Ile Val Leu Lys Ile Glu
Gly Glu Lys Tyr Thr 370 375 380cta gag aca ccc atc gca gat cac tcc
gaa ggc cta aac ctg gcg ttc 2157Leu Glu Thr Pro Ile Ala Asp His Ser
Glu Gly Leu Asn Leu Ala Phe385 390 395 400gat ctc atg gac cag cac
aac tgt ggt cct tcc caa ctg gaa atc acc 2205Asp Leu Met Asp Gln His
Asn Cys Gly Pro Ser Gln Leu Glu Ile Thr 405 410 415gca gtt gga cac
cgc gtg gtc cac ggc gga atc ttg ttc tcc gca ccg 2253Ala Val Gly His
Arg Val Val His Gly Gly Ile Leu Phe Ser Ala Pro 420 425 430gaa ctt
atc act gat gaa atc gtg gaa atg atc cgc gat ctc att cca 2301Glu Leu
Ile Thr Asp Glu Ile Val Glu Met Ile Arg Asp Leu Ile Pro 435 440
445ctc gca cca ctg cac aac cct gca aac gtt gac ggc att gat gtt gct
2349Leu Ala Pro Leu His Asn Pro Ala Asn Val Asp Gly Ile Asp Val Ala
450 455 460cga aaa att ctc ccc gat gtc cca cac gta gct gtc ttt gac
acc ggt 2397Arg Lys Ile Leu Pro Asp Val Pro His Val Ala Val Phe Asp
Thr Gly465 470 475 480ttc ttc cac tca ctt cca cca gca gct gcg ctg
tat gcc atc aac aag 2445Phe Phe His Ser Leu Pro Pro Ala Ala Ala Leu
Tyr Ala Ile Asn Lys 485 490 495gat gtc gca gct gaa cac gga atc agg
cgc tat ggt ttc cac ggc acc 2493Asp Val Ala Ala Glu His Gly Ile Arg
Arg Tyr Gly Phe His Gly Thr 500 505 510tcc cat gaa ttt gtg tcc aag
cgc gtg gtg gaa att ctg gaa aag ccc 2541Ser His Glu Phe Val Ser Lys
Arg Val Val Glu Ile Leu Glu Lys Pro 515 520 525acc gaa gac atc aac
acc atc acc ttc cac ctg ggc aac ggc gca tcc 2589Thr Glu Asp Ile Asn
Thr Ile Thr Phe His Leu Gly Asn Gly Ala Ser 530 535 540atg gct gct
gtt caa ggt ggc cgt gcg gta gat act tcc atg ggt atg 2637Met Ala Ala
Val Gln Gly Gly Arg Ala Val Asp Thr Ser Met Gly Met545 550 555
560aca cct ctc gcg ggc ctt gtc atg ggt acc cga agc ggt gac att gat
2685Thr Pro Leu Ala Gly Leu Val Met Gly Thr Arg Ser Gly Asp Ile Asp
565 570 575cca ggt atc gtc ttc cac ctt tcc cgc acc gct ggc atg agc
atc gat 2733Pro Gly Ile Val Phe His Leu Ser Arg Thr Ala Gly Met Ser
Ile Asp 580 585 590gag atc gat aat ctg ctg aac aaa aag tcg ggt gta
aag gga ctt tcc 2781Glu Ile Asp Asn Leu Leu Asn Lys Lys Ser Gly Val
Lys Gly Leu Ser 595 600 605ggt gtt aat gat ttc cgt gaa ctg cgg gaa
atg atc gac aac aat gat 2829Gly Val Asn Asp Phe Arg Glu Leu Arg Glu
Met Ile Asp Asn Asn Asp 610 615 620caa gat gcc tgg tcc gcg tac aac
att tac ata cac caa ctc cgc cgc 2877Gln Asp Ala Trp Ser Ala Tyr Asn
Ile Tyr Ile His Gln Leu Arg Arg625 630 635 640tac ctc ggt tcc tac
atg gtg gca ctg gga cgg gta gac acc atc gtg 2925Tyr Leu Gly Ser Tyr
Met Val Ala Leu Gly Arg Val Asp Thr Ile Val 645 650 655ttc acc gcc
ggt gtc ggt gaa aat gcc cag ttt gtc cgt gag gat gcc 2973Phe Thr Ala
Gly Val Gly Glu Asn Ala Gln Phe Val Arg Glu Asp Ala 660 665 670ttg
gca ggt ttg gaa atg tac gga att gag atc gat cca gag cgt aac 3021Leu
Ala Gly Leu Glu Met Tyr Gly Ile Glu Ile Asp Pro Glu Arg Asn 675 680
685gca ttg cca aac gat ggt cct cga ttg att tcc acc gat gcc tcc aag
3069Ala Leu Pro Asn Asp Gly Pro Arg Leu Ile Ser Thr Asp Ala Ser Lys
690 695 700gtg aag gtg ttt gtt att cca act aat gaa gag tta gct atc
gct agg 3117Val Lys Val Phe Val Ile Pro Thr Asn Glu Glu Leu Ala Ile
Ala Arg705 710 715 720tac gcg gtg aag ttc gct tagctctcct ggttaggatc
caccacaaat 3165Tyr Ala Val Lys Phe Ala 725cgctctgatc agcggttttg
tggtggattt ttgcgttttt aaggggtgaa actgcacgga 3225tccaccacag
atcccagttt tcctttggaa cgtggtggat ccttgccctg gagcttcaca
3285ggaatcgctt gttggcccct agacctcttg gggttgcgaa ttttcgtccc
caccgaacat 3345taaaaggccg gttttggtcg aaaatttgct ctaacacctt
gctattatgc gaatattcgt 3405tccatttcat cgaattccag caacccgtaa
cgagaagttg aacaggaaac ctgcagtaac 3465cccgcagaaa tcacatcagc
cccaattgtc ccaaaagtaa ctcccccaga atcgcttcta 3525agggcctaac
tcgcccaaag tcaaactagg ggacatcgca ctcctaaagg cccttaaatc
3585gccacctacc aaatagcccc aagtcaaaac agctagaacc aactcagtgg
ccgcacggca 3645ttcgccatat ccacaagtgc gtaacggtgg tgcgggaacg
gtgcagaacg tgcctgaatg 3705cggagtgcct cggagatgcc ggtgcgcagg
cctttttggg agaacgggta ttcaaacaaa 3765gggttcgcgg aggcggaagc
tttgagtttt cgctctcgaa gccagctgag gcctgccgac 3825atgatggcaa
ttttgatttg gttgaagcgg ggttcgtttg tggggatttc ggtgagtcgg
3885cgggcagccc gtcggatgcg ggattcactc aaattggagc tgaccagcaa
caagatggtg 3945gtgagggtgg ccattcggta gtgggtggat gattggggga
gtttgtctag ggcttggact 4005gcgagttcga tttggttttc ggccatgagt
tggcgggcga gcccgaacgc ggaggacacg 4065gtggtggggt tggttgccca
gacaagtgcg tagaggcgca gtgaatggaa gcggactacg 4125tgtgggtcgc
tggagatgtg ggaccaggtg tcgctgagtg attcgaaggc ggaggcgtcg
4185aggtcttcga aatct 420040329PRTCorynebacterium glutamicum 40Met
Ser Ala Glu Leu Phe Glu Asn Trp Leu Leu Lys Arg Ala Arg Ala1 5 10
15Glu His Ser His Ile Val Leu Pro Glu Gly Asp Asp Asp Arg Ile Leu
20 25 30Met Ala Ala His Gln Leu Leu Asp Gln Asp Ile Cys Asp Ile Thr
Ile 35 40 45Leu Gly Asp Pro Val Lys Ile Lys Glu Arg Ala Thr Glu Leu
Gly Leu 50 55 60His Leu Asn Thr Ala Tyr Leu Val Asn Pro Leu Thr Asp
Pro Arg Leu65 70 75 80Glu Glu Phe Ala Glu Gln Phe Ala Glu Leu Arg
Lys Ser Lys Ser Val 85 90 95Thr Ile Asp Glu Ala Arg Glu Ile Met Lys
Asp Ile Ser Tyr Phe Gly 100 105 110Thr Met Met Val His Asn Gly Asp
Ala Asp Gly Met Val Ser Gly Ala 115 120 125Ala Asn Thr Thr Ala His
Thr Ile Lys Pro Ser Phe Gln Ile Ile Lys 130 135 140Thr Val Pro Glu
Ala Ser Val Val Ser Ser Ile Phe Leu Met Val Leu145 150 155 160Arg
Gly Arg Leu Trp Ala
Phe Gly Asp Cys Ala Val Asn Pro Asn Pro 165 170 175Thr Ala Glu Gln
Leu Gly Glu Ile Ala Val Val Ser Ala Lys Thr Ala 180 185 190Ala Gln
Phe Gly Ile Asp Pro Arg Val Ala Ile Leu Ser Tyr Ser Thr 195 200
205Gly Asn Ser Gly Gly Gly Ser Asp Val Asp Arg Ala Ile Asp Ala Leu
210 215 220Ala Glu Ala Arg Arg Leu Asn Pro Glu Leu Cys Val Asp Gly
Pro Leu225 230 235 240Gln Phe Asp Ala Ala Val Asp Pro Gly Val Ala
Arg Lys Lys Met Pro 245 250 255Asp Ser Asp Val Ala Gly Gln Ala Asn
Val Phe Ile Phe Pro Asp Leu 260 265 270Glu Ala Gly Asn Ile Gly Tyr
Lys Thr Ala Gln Arg Thr Gly His Ala 275 280 285Leu Ala Val Gly Pro
Ile Leu Gln Gly Leu Asn Lys Pro Val Asn Asp 290 295 300Leu Ser Arg
Gly Ala Thr Val Pro Asp Ile Val Asn Thr Val Ala Ile305 310 315
320Thr Ala Ile Gln Ala Gly Gly Arg Ser 32541397PRTCorynebacterium
glutamicum 41Met Ala Leu Ala Leu Val Leu Asn Ser Gly Ser Ser Ser
Ile Lys Phe1 5 10 15Gln Leu Val Asn Pro Glu Asn Ser Ala Ile Asp Glu
Pro Tyr Val Ser 20 25 30Gly Leu Val Glu Gln Ile Gly Glu Pro Asn Gly
Arg Ile Val Leu Lys 35 40 45Ile Glu Gly Glu Lys Tyr Thr Leu Glu Thr
Pro Ile Ala Asp His Ser 50 55 60Glu Gly Leu Asn Leu Ala Phe Asp Leu
Met Asp Gln His Asn Cys Gly65 70 75 80Pro Ser Gln Leu Glu Ile Thr
Ala Val Gly His Arg Val Val His Gly 85 90 95Gly Ile Leu Phe Ser Ala
Pro Glu Leu Ile Thr Asp Glu Ile Val Glu 100 105 110Met Ile Arg Asp
Leu Ile Pro Leu Ala Pro Leu His Asn Pro Ala Asn 115 120 125Val Asp
Gly Ile Asp Val Ala Arg Lys Ile Leu Pro Asp Val Pro His 130 135
140Val Ala Val Phe Asp Thr Gly Phe Phe His Ser Leu Pro Pro Ala
Ala145 150 155 160Ala Leu Tyr Ala Ile Asn Lys Asp Val Ala Ala Glu
His Gly Ile Arg 165 170 175Arg Tyr Gly Phe His Gly Thr Ser His Glu
Phe Val Ser Lys Arg Val 180 185 190Val Glu Ile Leu Glu Lys Pro Thr
Glu Asp Ile Asn Thr Ile Thr Phe 195 200 205His Leu Gly Asn Gly Ala
Ser Met Ala Ala Val Gln Gly Gly Arg Ala 210 215 220Val Asp Thr Ser
Met Gly Met Thr Pro Leu Ala Gly Leu Val Met Gly225 230 235 240Thr
Arg Ser Gly Asp Ile Asp Pro Gly Ile Val Phe His Leu Ser Arg 245 250
255Thr Ala Gly Met Ser Ile Asp Glu Ile Asp Asn Leu Leu Asn Lys Lys
260 265 270Ser Gly Val Lys Gly Leu Ser Gly Val Asn Asp Phe Arg Glu
Leu Arg 275 280 285Glu Met Ile Asp Asn Asn Asp Gln Asp Ala Trp Ser
Ala Tyr Asn Ile 290 295 300Tyr Ile His Gln Leu Arg Arg Tyr Leu Gly
Ser Tyr Met Val Ala Leu305 310 315 320Gly Arg Val Asp Thr Ile Val
Phe Thr Ala Gly Val Gly Glu Asn Ala 325 330 335Gln Phe Val Arg Glu
Asp Ala Leu Ala Gly Leu Glu Met Tyr Gly Ile 340 345 350Glu Ile Asp
Pro Glu Arg Asn Ala Leu Pro Asn Asp Gly Pro Arg Leu 355 360 365Ile
Ser Thr Asp Ala Ser Lys Val Lys Val Phe Val Ile Pro Thr Asn 370 375
380Glu Glu Leu Ala Ile Ala Arg Tyr Ala Val Lys Phe Ala385 390
395423780DNACorynebacterium glutamicumCDS(996)..(2732) 42taatgaggaa
aaccgaaccc caccagaaga attccaacag cgcaccacca atgatcgggc 60ctgccgcagc
gccaagaatt gccacggaac cccaaatacc aattgcagtg ttgcgctcac
120gctcatcctc aaacgtaatg cggatcagag ccaaggttgc aggcatcatc
gttgccgcac 180cgatgccaag gaaagctctc gcagcaacaa gagcccacgc
agttggagca aacgcagcac 240caagtgaagc gattccgaaa atgctcaagc
ccatgaggaa catccggcgg tggccgattt 300tgtcacccaa agtgccggta
cccaaaagaa ggcccgccat gagcagggga tatgcgttga 360tgatccacaa
cgcttgggtt tcggtggctg cgagctgttc acgcagcaga gggagtgcgg
420tgtagagaat cgagttgtct acaccgatca gaaagagacc accgctgata
acggcgagga 480aagcccaacg ttgggttttc gtaggcgctt gcgcctgtaa
ggtttctgaa gtcatggatc 540gtaactgtaa cgaatggtcg gtacagttac
aactcttttg ttggtgtttt agaccacggc 600gctgtgtggc gatttaagac
gtcggaaatc gtaggggact gtcagcgtgg gtcgggttct 660ttgaggcgct
tagaggcgat tctgtgaggt cactttttgt ggggtcgggg tctaaatttg
720gccagttttc gaggcgacca gacaggcgtg cccacgatgt ttaaataggc
gatcggtggg 780catctgtgtt tggtttcgac gggctgaaac caaaccagac
tgcccagcaa cgacggaaat 840cccaaaagtg ggcatccctg tttggtaccg
agtacccacc cgggcctgaa actccctggc 900aggcgggcga agcgtggcaa
caactggaat ttaagagcac aattgaagtc gcaccaagtt 960aggcaacaca
atagccataa cgttgaggag ttcag atg gca cac agc tac gca 1013 Met Ala
His Ser Tyr Ala 1 5gaa caa tta att gac act ttg gaa gct caa ggt gtg
aag cga att tat 1061Glu Gln Leu Ile Asp Thr Leu Glu Ala Gln Gly Val
Lys Arg Ile Tyr 10 15 20ggt ttg gtg ggt gac agc ctt aat ccg atc gtg
gat gct gtc cgc caa 1109Gly Leu Val Gly Asp Ser Leu Asn Pro Ile Val
Asp Ala Val Arg Gln 25 30 35tca gat att gag tgg gtg cac gtt cga aat
gag gaa gcg gcg gcg ttt 1157Ser Asp Ile Glu Trp Val His Val Arg Asn
Glu Glu Ala Ala Ala Phe 40 45 50gca gcc ggt gcg gaa tcg ttg atc act
ggg gag ctg gca gta tgt gct 1205Ala Ala Gly Ala Glu Ser Leu Ile Thr
Gly Glu Leu Ala Val Cys Ala55 60 65 70gct tct tgt ggt cct gga aac
aca cac ctg att cag ggt ctt tat gat 1253Ala Ser Cys Gly Pro Gly Asn
Thr His Leu Ile Gln Gly Leu Tyr Asp 75 80 85tcg cat cga aat ggt gcg
aag gtg ttg gcc atc gct agc cat att ccg 1301Ser His Arg Asn Gly Ala
Lys Val Leu Ala Ile Ala Ser His Ile Pro 90 95 100agt gcc cag att
ggt tcg acg ttc ttc cag gaa acg cat ccg gag att 1349Ser Ala Gln Ile
Gly Ser Thr Phe Phe Gln Glu Thr His Pro Glu Ile 105 110 115ttg ttt
aag gaa tgc tct ggt tac tgc gag atg gtg aat ggt ggt gag 1397Leu Phe
Lys Glu Cys Ser Gly Tyr Cys Glu Met Val Asn Gly Gly Glu 120 125
130cag ggt gaa cgc att ttg cat cac gcg att cag tcc acc atg gcg ggt
1445Gln Gly Glu Arg Ile Leu His His Ala Ile Gln Ser Thr Met Ala
Gly135 140 145 150aaa ggt gtg tcg gtg gta gtg att cct ggt gat atc
gct aag gaa gac 1493Lys Gly Val Ser Val Val Val Ile Pro Gly Asp Ile
Ala Lys Glu Asp 155 160 165gca ggt gac ggt act tat tcc aat tcc act
att tct tct ggc act cct 1541Ala Gly Asp Gly Thr Tyr Ser Asn Ser Thr
Ile Ser Ser Gly Thr Pro 170 175 180gtg gtg ttc ccg gat cct act gag
gct gca gcg ctg gtg gag gcg att 1589Val Val Phe Pro Asp Pro Thr Glu
Ala Ala Ala Leu Val Glu Ala Ile 185 190 195aac aac gct aag tct gtc
act ttg ttc tgc ggt gcg ggc gtg aag aat 1637Asn Asn Ala Lys Ser Val
Thr Leu Phe Cys Gly Ala Gly Val Lys Asn 200 205 210gct cgc gcg cag
gtg ttg gag ttg gcg gag aag att aaa tca ccg atc 1685Ala Arg Ala Gln
Val Leu Glu Leu Ala Glu Lys Ile Lys Ser Pro Ile215 220 225 230ggg
cat gcg ctg ggt ggt aag cag tac atc cag cat gag aat ccg ttt 1733Gly
His Ala Leu Gly Gly Lys Gln Tyr Ile Gln His Glu Asn Pro Phe 235 240
245gag gtc ggc atg tct ggc ctg ctt ggt tac ggc gcc tgc gtg gat gcg
1781Glu Val Gly Met Ser Gly Leu Leu Gly Tyr Gly Ala Cys Val Asp Ala
250 255 260tcc aat gag gcg gat ctg ctg att cta ttg ggt acg gat ttc
cct tat 1829Ser Asn Glu Ala Asp Leu Leu Ile Leu Leu Gly Thr Asp Phe
Pro Tyr 265 270 275tct gat ttc ctt cct aaa gac aac gtt gcc cag gtg
gat atc aac ggt 1877Ser Asp Phe Leu Pro Lys Asp Asn Val Ala Gln Val
Asp Ile Asn Gly 280 285 290gcg cac att ggt cga cgt acc acg gtg aag
tat ccg gtg acc ggt gat 1925Ala His Ile Gly Arg Arg Thr Thr Val Lys
Tyr Pro Val Thr Gly Asp295 300 305 310gtt gct gca aca atc gaa aat
att ttg cct cat gtg aag gaa aaa aca 1973Val Ala Ala Thr Ile Glu Asn
Ile Leu Pro His Val Lys Glu Lys Thr 315 320 325gat cgt tcc ttc ctt
gat cgg atg ctc aag gca cac gag cgt aag ttg 2021Asp Arg Ser Phe Leu
Asp Arg Met Leu Lys Ala His Glu Arg Lys Leu 330 335 340agc tcg gtg
gta gag acg tac aca cat aac gtc gag aag cat gtg cct 2069Ser Ser Val
Val Glu Thr Tyr Thr His Asn Val Glu Lys His Val Pro 345 350 355att
cac cct gaa tac gtt gcc tct att ttg aac gag ctg gcg gat aag 2117Ile
His Pro Glu Tyr Val Ala Ser Ile Leu Asn Glu Leu Ala Asp Lys 360 365
370gat gcg gtg ttt act gtg gat acc ggc atg tgc aat gtg tgg cat gcg
2165Asp Ala Val Phe Thr Val Asp Thr Gly Met Cys Asn Val Trp His
Ala375 380 385 390agg tac atc gag aat ccg gag gga acg cgc gac ttt
gtg ggt tca ttc 2213Arg Tyr Ile Glu Asn Pro Glu Gly Thr Arg Asp Phe
Val Gly Ser Phe 395 400 405cgc cac ggc acg atg gct aat gcg ttg cct
cat gcg att ggt gcg caa 2261Arg His Gly Thr Met Ala Asn Ala Leu Pro
His Ala Ile Gly Ala Gln 410 415 420agt gtt gat cga aac cgc cag gtg
atc gcg atg tgt ggc gat ggt ggt 2309Ser Val Asp Arg Asn Arg Gln Val
Ile Ala Met Cys Gly Asp Gly Gly 425 430 435ttg ggc atg ctg ctg ggt
gag ctt ctg acc gtt aag ctg cac caa ctt 2357Leu Gly Met Leu Leu Gly
Glu Leu Leu Thr Val Lys Leu His Gln Leu 440 445 450ccg ctg aag gct
gtg gtg ttt aac aac agt tct ttg ggc atg gtg aag 2405Pro Leu Lys Ala
Val Val Phe Asn Asn Ser Ser Leu Gly Met Val Lys455 460 465 470ttg
gag atg ctc gtg gag gga cag cca gaa ttt ggt act gac cat gag 2453Leu
Glu Met Leu Val Glu Gly Gln Pro Glu Phe Gly Thr Asp His Glu 475 480
485gaa gtg aat ttc gca gag att gcg gcg gct gcg ggt atc aaa tcg gta
2501Glu Val Asn Phe Ala Glu Ile Ala Ala Ala Ala Gly Ile Lys Ser Val
490 495 500cgc atc acc gat ccg aag aaa gtt cgc gag cag cta gct gag
gca ttg 2549Arg Ile Thr Asp Pro Lys Lys Val Arg Glu Gln Leu Ala Glu
Ala Leu 505 510 515gca tat cct gga cct gta ctg atc gat atc gtc acg
gat cct aat gcg 2597Ala Tyr Pro Gly Pro Val Leu Ile Asp Ile Val Thr
Asp Pro Asn Ala 520 525 530ctg tcg atc cca cca acc atc acg tgg gaa
cag gtc atg gga ttc agc 2645Leu Ser Ile Pro Pro Thr Ile Thr Trp Glu
Gln Val Met Gly Phe Ser535 540 545 550aag gcg gcc acc cga acc gtc
ttt ggt gga gga gta gga gcg atg atc 2693Lys Ala Ala Thr Arg Thr Val
Phe Gly Gly Gly Val Gly Ala Met Ile 555 560 565gat ctg gcc cgt tcg
aac ata agg aat att cct act cca tgatgattga 2742Asp Leu Ala Arg Ser
Asn Ile Arg Asn Ile Pro Thr Pro 570 575tacacctgct gttctcattg
accgcgagcg cttaactgcc aacatttcca ggatggcagc 2802tcacgccggt
gcccatgaga ttgccctgcg tccgcatgtg aaaacgcaca aaatcattga
2862aattgcgcag atgcaggtcg acgccggtgc ccgagggatc acctgcgcaa
ccattggcga 2922ggcggaaatt tttgccggcg caggttttac ggacatcttt
attgcatatc cgctgtatct 2982aaccgatcat gcagtgcaac gcctgaacgc
gatccccgga gaaatttcca ttggcgtgga 3042ttcggtagag atggcacagg
cgacggcggg tttgcgggaa gatatcaagg ctctgattga 3102agtggattcg
ggacatcgta gaagtggagt cacggcgact gcttcagaat tgagtcagat
3162ccgcgaggcg ctgggcagca ggtatgcagg agtgtttact tttcctgggc
attcttatgg 3222cccgggaaat ggtgagcagg cagcagctga tgagcttcag
gctctaaaca acagcgtcca 3282gcgacttgct ggcggcctga cttctggcgg
ttcctcgccg tctgcgcagt ttacagacgc 3342aatcgatgag atgcgaccag
gcgtgtatgt gtttaacgat tcccagcaga tcacctcggg 3402agcatgcact
gagaagcagg tggcaatgac ggtgctgtct actgtggtca gccgaaatgt
3462gtcagatcgt cggatcattt tggatgcggg atccaaaatc ctcagcactg
ataaaccagc 3522atggattgat ggcaatggtt ttgttctggg gaatcctgaa
gcccgaatct ctgctttgtc 3582ggagcatcac gcaaccattt tctggccaga
taaagtgcta cttccagtaa tcggggagca 3642gctcaacatc gtgcccaacc
atgcctgcaa cgtgattaat ttggtggatg aggtctacgt 3702tcgggaagcc
gatggcactt tccgtacctg gaaggtagtt gcccgcggca gaaacaatta
3762gggaaacctc ttgacctt 378043579PRTCorynebacterium glutamicum
43Met Ala His Ser Tyr Ala Glu Gln Leu Ile Asp Thr Leu Glu Ala Gln1
5 10 15Gly Val Lys Arg Ile Tyr Gly Leu Val Gly Asp Ser Leu Asn Pro
Ile 20 25 30Val Asp Ala Val Arg Gln Ser Asp Ile Glu Trp Val His Val
Arg Asn 35 40 45Glu Glu Ala Ala Ala Phe Ala Ala Gly Ala Glu Ser Leu
Ile Thr Gly 50 55 60Glu Leu Ala Val Cys Ala Ala Ser Cys Gly Pro Gly
Asn Thr His Leu65 70 75 80Ile Gln Gly Leu Tyr Asp Ser His Arg Asn
Gly Ala Lys Val Leu Ala 85 90 95Ile Ala Ser His Ile Pro Ser Ala Gln
Ile Gly Ser Thr Phe Phe Gln 100 105 110Glu Thr His Pro Glu Ile Leu
Phe Lys Glu Cys Ser Gly Tyr Cys Glu 115 120 125Met Val Asn Gly Gly
Glu Gln Gly Glu Arg Ile Leu His His Ala Ile 130 135 140Gln Ser Thr
Met Ala Gly Lys Gly Val Ser Val Val Val Ile Pro Gly145 150 155
160Asp Ile Ala Lys Glu Asp Ala Gly Asp Gly Thr Tyr Ser Asn Ser Thr
165 170 175Ile Ser Ser Gly Thr Pro Val Val Phe Pro Asp Pro Thr Glu
Ala Ala 180 185 190Ala Leu Val Glu Ala Ile Asn Asn Ala Lys Ser Val
Thr Leu Phe Cys 195 200 205Gly Ala Gly Val Lys Asn Ala Arg Ala Gln
Val Leu Glu Leu Ala Glu 210 215 220Lys Ile Lys Ser Pro Ile Gly His
Ala Leu Gly Gly Lys Gln Tyr Ile225 230 235 240Gln His Glu Asn Pro
Phe Glu Val Gly Met Ser Gly Leu Leu Gly Tyr 245 250 255Gly Ala Cys
Val Asp Ala Ser Asn Glu Ala Asp Leu Leu Ile Leu Leu 260 265 270Gly
Thr Asp Phe Pro Tyr Ser Asp Phe Leu Pro Lys Asp Asn Val Ala 275 280
285Gln Val Asp Ile Asn Gly Ala His Ile Gly Arg Arg Thr Thr Val Lys
290 295 300Tyr Pro Val Thr Gly Asp Val Ala Ala Thr Ile Glu Asn Ile
Leu Pro305 310 315 320His Val Lys Glu Lys Thr Asp Arg Ser Phe Leu
Asp Arg Met Leu Lys 325 330 335Ala His Glu Arg Lys Leu Ser Ser Val
Val Glu Thr Tyr Thr His Asn 340 345 350Val Glu Lys His Val Pro Ile
His Pro Glu Tyr Val Ala Ser Ile Leu 355 360 365Asn Glu Leu Ala Asp
Lys Asp Ala Val Phe Thr Val Asp Thr Gly Met 370 375 380Cys Asn Val
Trp His Ala Arg Tyr Ile Glu Asn Pro Glu Gly Thr Arg385 390 395
400Asp Phe Val Gly Ser Phe Arg His Gly Thr Met Ala Asn Ala Leu Pro
405 410 415His Ala Ile Gly Ala Gln Ser Val Asp Arg Asn Arg Gln Val
Ile Ala 420 425 430Met Cys Gly Asp Gly Gly Leu Gly Met Leu Leu Gly
Glu Leu Leu Thr 435 440 445Val Lys Leu His Gln Leu Pro Leu Lys Ala
Val Val Phe Asn Asn Ser 450 455 460Ser Leu Gly Met Val Lys Leu Glu
Met Leu Val Glu Gly Gln Pro Glu465 470 475 480Phe Gly Thr Asp His
Glu Glu Val Asn Phe Ala Glu Ile Ala Ala Ala 485 490 495Ala Gly Ile
Lys Ser Val Arg Ile Thr Asp Pro Lys Lys Val Arg Glu 500 505 510Gln
Leu Ala Glu Ala Leu Ala Tyr Pro Gly Pro Val Leu Ile Asp Ile 515 520
525Val Thr Asp Pro Asn Ala Leu Ser Ile Pro Pro Thr Ile Thr Trp Glu
530 535 540Gln Val Met Gly Phe Ser Lys Ala Ala Thr Arg Thr Val Phe
Gly Gly545 550 555 560Gly Val Gly Ala Met Ile Asp Leu Ala Arg Ser
Asn Ile Arg Asn Ile 565 570 575Pro Thr Pro443600DNACorynebacterium
glutamicumCDS(1037)..(2542) 44gaagcgctac ggacttcgcg ccggcgtcga
cagcaatgcg tccagcatcc aagtgagtat 60ggtgctcatc atcaatacca acgcggaact
tcaccgtcac cggaatgtcc gtgccttccg 120tagccttcac agccgcggaa
acgatgtttt caaacaaacg gcgcttgtaa ggaatcgcag
180aaccgccacc ccggcgcgtg acctttggaa ccgggcagcc aaagttcata
tcaatatgat 240ccgccaagtt ttcatcaacg atcatcttcg ccgcttcgta
ggtgtacttc gggtcaaccg 300tgtacagctg caagcttcgg ggattttcat
ccggcgcgaa ggtggtcatg tgcatggttt 360tctcattgcg ctcaacaaga
gcacgcgcag tcaccatttc acagacgtac agccccgaga 420ttgttcccgt
gcgttgcatt tcctgttcac ggcacagcgt gcggaaagca acgttggtta
480caccagccat gggggctaga accacagggg aggcaaggtc aaaggggccg
atttttaaag 540tcacctaact attgtccccc gtgaatcagg ttgggcaaaa
tatttgaagc aaattgtgag 600cagggcgcaa ctaggaaagt ggtgtgcttt
cactttttgg gggctggggt tgggttaagc 660ttcgcgggct ctagggttgg
tctgagcttt attcctgggc tttgggaggc ttgcaaacag 720ggggcatgca
aatttggggg taatgctggg ccttgaaatc ccactatcac agatagtatt
780cgggcatttc ctgtcacgat ggtttatcct tgggacacaa catcaaagtg
gggtacatca 840tatgcttccg gttgaagtga cctatctgaa aagattggtc
gaaccttgaa gcaatggtgt 900gaactgcgtt aacgaatttt gtcggacgtt
aaaatggtcg cattctgctt gctgaagtgg 960cacacctatg tgttctgctt
gggtatagca gtgcgggaaa aatttgaaaa agtccgatta 1020cctgaggagg tattca
atg tct gat cgc att gct tca gaa aag ctg cgc tcc 1072 Met Ser Asp
Arg Ile Ala Ser Glu Lys Leu Arg Ser 1 5 10aag ctc atg tcc gcc gac
gag gcg gca cag ttt gtt aac cac ggt gac 1120Lys Leu Met Ser Ala Asp
Glu Ala Ala Gln Phe Val Asn His Gly Asp 15 20 25aag gtt ggt ttc tcc
ggc ttc acc ggc gct ggc tac cca aag gca ctg 1168Lys Val Gly Phe Ser
Gly Phe Thr Gly Ala Gly Tyr Pro Lys Ala Leu 30 35 40cct acg gca atc
gct aac cgg gct aaa gaa gca cac ggt gca ggc aac 1216Pro Thr Ala Ile
Ala Asn Arg Ala Lys Glu Ala His Gly Ala Gly Asn45 50 55 60gac tac
gca atc gac ctg ttc act ggc gca tcg acc gcc cct gac tgc 1264Asp Tyr
Ala Ile Asp Leu Phe Thr Gly Ala Ser Thr Ala Pro Asp Cys 65 70 75gat
ggc gta ctt gca gaa gct gac gct atc cgc tgg cgc atg cca tac 1312Asp
Gly Val Leu Ala Glu Ala Asp Ala Ile Arg Trp Arg Met Pro Tyr 80 85
90gca tct gat cca atc atg cgt aac aag atc aac tcc ggc tcc atg gga
1360Ala Ser Asp Pro Ile Met Arg Asn Lys Ile Asn Ser Gly Ser Met Gly
95 100 105tac tcc gat atc cac ctg tcc cac tcc ggc cag cag gtt gaa
gag ggc 1408Tyr Ser Asp Ile His Leu Ser His Ser Gly Gln Gln Val Glu
Glu Gly 110 115 120ttc ttc ggc cag ctc aac gta gct gtc att gaa atc
acc cgc atc act 1456Phe Phe Gly Gln Leu Asn Val Ala Val Ile Glu Ile
Thr Arg Ile Thr125 130 135 140gaa gag ggc tac atc atc cct tct tcc
tcc gtg ggt aac aac gtt gag 1504Glu Glu Gly Tyr Ile Ile Pro Ser Ser
Ser Val Gly Asn Asn Val Glu 145 150 155tgg ctc aac gct gca gag aag
gtc atc ctc gag gtt aac tct tgg cag 1552Trp Leu Asn Ala Ala Glu Lys
Val Ile Leu Glu Val Asn Ser Trp Gln 160 165 170tct gaa gac ctc gaa
ggt atg cac gac atc tgg tct gtt cct gcc ctg 1600Ser Glu Asp Leu Glu
Gly Met His Asp Ile Trp Ser Val Pro Ala Leu 175 180 185cca aac cgc
att gcc gtg cca atc aac aag cca ggc gac cgc atc ggt 1648Pro Asn Arg
Ile Ala Val Pro Ile Asn Lys Pro Gly Asp Arg Ile Gly 190 195 200aag
acc tac atc gag ttc gac acc gac aag gtt gtt gct gtt gtt gag 1696Lys
Thr Tyr Ile Glu Phe Asp Thr Asp Lys Val Val Ala Val Val Glu205 210
215 220acc aac acc gca gac cgc aac gca cca ttc aag cct gtc gac gac
atc 1744Thr Asn Thr Ala Asp Arg Asn Ala Pro Phe Lys Pro Val Asp Asp
Ile 225 230 235tct aag aag atc gct ggc aac ttc ctc gac ttc ctg gaa
agc gaa gtt 1792Ser Lys Lys Ile Ala Gly Asn Phe Leu Asp Phe Leu Glu
Ser Glu Val 240 245 250gct gca ggt cgc ctg tcc tac gac ggc tac atc
atg cag tcc ggc gtg 1840Ala Ala Gly Arg Leu Ser Tyr Asp Gly Tyr Ile
Met Gln Ser Gly Val 255 260 265ggc aac gtg cca aac gcg gtg atg gca
ggc ctg ctg gaa tcc aag ttt 1888Gly Asn Val Pro Asn Ala Val Met Ala
Gly Leu Leu Glu Ser Lys Phe 270 275 280gag aac atc cag gcc tac acc
gaa gtt atc cag gac ggc atg gtg gac 1936Glu Asn Ile Gln Ala Tyr Thr
Glu Val Ile Gln Asp Gly Met Val Asp285 290 295 300ctc atc gac gcc
ggc aag atg acc gtt gca tcc gca act tcc ttc tcc 1984Leu Ile Asp Ala
Gly Lys Met Thr Val Ala Ser Ala Thr Ser Phe Ser 305 310 315ctg tct
cct gag tac gca gag aag atg aac aac gag gct aag cgt tac 2032Leu Ser
Pro Glu Tyr Ala Glu Lys Met Asn Asn Glu Ala Lys Arg Tyr 320 325
330cgc gag tcc att atc ctg cgc cca cag cag atc tct aac cac cca gag
2080Arg Glu Ser Ile Ile Leu Arg Pro Gln Gln Ile Ser Asn His Pro Glu
335 340 345gtc atc cgc cgc gtt ggc ctg atc gcc acc aac ggt ctc atc
gag gct 2128Val Ile Arg Arg Val Gly Leu Ile Ala Thr Asn Gly Leu Ile
Glu Ala 350 355 360gac att tac ggc aac gtc aac tcc acc aac gtt tct
ggc tcc cgc gtc 2176Asp Ile Tyr Gly Asn Val Asn Ser Thr Asn Val Ser
Gly Ser Arg Val365 370 375 380atg aac ggc atc ggc ggc tcc ggc gac
ttc acc cgt aac ggc tac atc 2224Met Asn Gly Ile Gly Gly Ser Gly Asp
Phe Thr Arg Asn Gly Tyr Ile 385 390 395tcc agc ttc atc acc cct tca
gag gca aag ggc ggc gca atc tct gcg 2272Ser Ser Phe Ile Thr Pro Ser
Glu Ala Lys Gly Gly Ala Ile Ser Ala 400 405 410atc gtt cct ttc gca
tcc cac atc gac cac acc gag cac gat gtc atg 2320Ile Val Pro Phe Ala
Ser His Ile Asp His Thr Glu His Asp Val Met 415 420 425gtt gtt atc
tct gag tac ggt tac gca gac ctt cgt ggt ctg gct cca 2368Val Val Ile
Ser Glu Tyr Gly Tyr Ala Asp Leu Arg Gly Leu Ala Pro 430 435 440cgt
gag cgc gtt gcc aag atg atc ggc ctg gct cac cct gat tac cgc 2416Arg
Glu Arg Val Ala Lys Met Ile Gly Leu Ala His Pro Asp Tyr Arg445 450
455 460cca ctg ctc gag gag tac tac gct cgc gca acc tcc ggt gac aac
aag 2464Pro Leu Leu Glu Glu Tyr Tyr Ala Arg Ala Thr Ser Gly Asp Asn
Lys 465 470 475tac atg cag acc cct cat gat ctt gca acc gcg ttt gat
ttc cac atc 2512Tyr Met Gln Thr Pro His Asp Leu Ala Thr Ala Phe Asp
Phe His Ile 480 485 490aac ctg gct aag aac ggc tcc atg aag gca
taagtttttt cttggtttag 2562Asn Leu Ala Lys Asn Gly Ser Met Lys Ala
495 500aaaccgccgc ctcgacaaca tttcgaggcg gcggtttctt ttattacctg
ggttttgagc 2622gttaaattag accaggtcag gctagtgttt ggtagctaat
tgagggcgat tttaataagg 2682ccggtgccat gtactaatat ggtctgagtt
gggcctatag ctcagttggt agagctacgg 2742acttttaatc cgcaggtctt
gggttcgagt cccaatgggc ccacatctta agtacccctg 2802ttttggagaa
tgctccgagc caggggtact tttcttttcc tcacacacag tagctgctga
2862gaaaaatgaa gaccttttgt taggttggga gtatgaccaa cccatacgag
gccttcatac 2922cgctcaagca tcgtacgggg attgaacccg agcacacctt
ttgggaatgg gaaaacaaaa 2982gggttcacat tgcaaggaga cgtcgagaag
cgcccgtccg cgttatcgtg gtgcatgggc 3042taggcaccca tagtggcgcc
ctctggcccc tcgtcgcggc cattgagggc gcggacctcg 3102ccgcgatcga
cctgcctaaa actccgcttt acgacgattg gctgcgcctt ttagaatctt
3162tcatctcttc cgaagacgac ggtcggccac tcatcctgat cggtgcaggc
accggaggct 3222tgctttgcgc agaagctgca caccgcacag gactggtcgc
acacgtcatt gccacctgcc 3282tgctcaaccc ctccgaccag ccgacgcgcc
gggcactgtt caggttttca ccgctgactc 3342ggttgatcca aggccgcttg
cgcaaccgcg aaattcccgt gaccagagtg ttgaacttca 3402gcaaaatcag
ccgcagccca gccctgagca aattgtgcgc ggccgatgaa tttagcggag
3462catccaaaat aacctggggt ttcctcgcgt catatgtgca acacaaggcc
aaactgggtg 3522cagttcccgt cactctgatg caccctgacc acgaccttct
gactcccgtt gagctcagtc 3582tgcgtacgct ttcgcgcc
360045502PRTCorynebacterium glutamicum 45Met Ser Asp Arg Ile Ala
Ser Glu Lys Leu Arg Ser Lys Leu Met Ser1 5 10 15Ala Asp Glu Ala Ala
Gln Phe Val Asn His Gly Asp Lys Val Gly Phe 20 25 30Ser Gly Phe Thr
Gly Ala Gly Tyr Pro Lys Ala Leu Pro Thr Ala Ile 35 40 45Ala Asn Arg
Ala Lys Glu Ala His Gly Ala Gly Asn Asp Tyr Ala Ile 50 55 60Asp Leu
Phe Thr Gly Ala Ser Thr Ala Pro Asp Cys Asp Gly Val Leu65 70 75
80Ala Glu Ala Asp Ala Ile Arg Trp Arg Met Pro Tyr Ala Ser Asp Pro
85 90 95Ile Met Arg Asn Lys Ile Asn Ser Gly Ser Met Gly Tyr Ser Asp
Ile 100 105 110His Leu Ser His Ser Gly Gln Gln Val Glu Glu Gly Phe
Phe Gly Gln 115 120 125Leu Asn Val Ala Val Ile Glu Ile Thr Arg Ile
Thr Glu Glu Gly Tyr 130 135 140Ile Ile Pro Ser Ser Ser Val Gly Asn
Asn Val Glu Trp Leu Asn Ala145 150 155 160Ala Glu Lys Val Ile Leu
Glu Val Asn Ser Trp Gln Ser Glu Asp Leu 165 170 175Glu Gly Met His
Asp Ile Trp Ser Val Pro Ala Leu Pro Asn Arg Ile 180 185 190Ala Val
Pro Ile Asn Lys Pro Gly Asp Arg Ile Gly Lys Thr Tyr Ile 195 200
205Glu Phe Asp Thr Asp Lys Val Val Ala Val Val Glu Thr Asn Thr Ala
210 215 220Asp Arg Asn Ala Pro Phe Lys Pro Val Asp Asp Ile Ser Lys
Lys Ile225 230 235 240Ala Gly Asn Phe Leu Asp Phe Leu Glu Ser Glu
Val Ala Ala Gly Arg 245 250 255Leu Ser Tyr Asp Gly Tyr Ile Met Gln
Ser Gly Val Gly Asn Val Pro 260 265 270Asn Ala Val Met Ala Gly Leu
Leu Glu Ser Lys Phe Glu Asn Ile Gln 275 280 285Ala Tyr Thr Glu Val
Ile Gln Asp Gly Met Val Asp Leu Ile Asp Ala 290 295 300Gly Lys Met
Thr Val Ala Ser Ala Thr Ser Phe Ser Leu Ser Pro Glu305 310 315
320Tyr Ala Glu Lys Met Asn Asn Glu Ala Lys Arg Tyr Arg Glu Ser Ile
325 330 335Ile Leu Arg Pro Gln Gln Ile Ser Asn His Pro Glu Val Ile
Arg Arg 340 345 350Val Gly Leu Ile Ala Thr Asn Gly Leu Ile Glu Ala
Asp Ile Tyr Gly 355 360 365Asn Val Asn Ser Thr Asn Val Ser Gly Ser
Arg Val Met Asn Gly Ile 370 375 380Gly Gly Ser Gly Asp Phe Thr Arg
Asn Gly Tyr Ile Ser Ser Phe Ile385 390 395 400Thr Pro Ser Glu Ala
Lys Gly Gly Ala Ile Ser Ala Ile Val Pro Phe 405 410 415Ala Ser His
Ile Asp His Thr Glu His Asp Val Met Val Val Ile Ser 420 425 430Glu
Tyr Gly Tyr Ala Asp Leu Arg Gly Leu Ala Pro Arg Glu Arg Val 435 440
445Ala Lys Met Ile Gly Leu Ala His Pro Asp Tyr Arg Pro Leu Leu Glu
450 455 460Glu Tyr Tyr Ala Arg Ala Thr Ser Gly Asp Asn Lys Tyr Met
Gln Thr465 470 475 480Pro His Asp Leu Ala Thr Ala Phe Asp Phe His
Ile Asn Leu Ala Lys 485 490 495Asn Gly Ser Met Lys Ala
500463423DNACorynebacterium glutamicumCDS(1)..(3420) 46gtg tcg act
cac aca tct tca acg ctt cca gca ttc aaa aag atc ttg 48Val Ser Thr
His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu1 5 10 15gta gca
aac cgc ggc gaa atc gcg gtc cgt gct ttc cgt gca gca ctc 96Val Ala
Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu 20 25 30gaa
acc ggt gca gcc acg gta gct att tac ccc cgt gaa gat cgg gga 144Glu
Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly 35 40
45tca ttc cac cgc tct ttt gct tct gaa gct gtc cgc att ggt acc gaa
192Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60ggc tca cca gtc aag gcg tac ctg gac atc gat gaa att atc ggt
gca 240Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly
Ala65 70 75 80gct aaa aaa gtt aaa gca gat gcc att tac ccg gga tac
ggc ttc ctg 288Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr
Gly Phe Leu 85 90 95tct gaa aat gcc cag ctt gcc cgc gag tgt gcg gaa
aac ggc att act 336Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu
Asn Gly Ile Thr 100 105 110ttt att ggc cca acc cca gag gtt ctt gat
ctc acc ggt gat aag tct 384Phe Ile Gly Pro Thr Pro Glu Val Leu Asp
Leu Thr Gly Asp Lys Ser 115 120 125cgc gcg gta acc gcc gcg aag aag
gct ggt ctg cca gtt ttg gcg gaa 432Arg Ala Val Thr Ala Ala Lys Lys
Ala Gly Leu Pro Val Leu Ala Glu 130 135 140tcc acc ccg agc aaa aac
atc gat gag atc gtt aaa agc gct gaa ggc 480Ser Thr Pro Ser Lys Asn
Ile Asp Glu Ile Val Lys Ser Ala Glu Gly145 150 155 160cag act tac
ccc atc ttt gtg aag gca gtt gcc ggt ggt ggc gga cgc 528Gln Thr Tyr
Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg 165 170 175ggt
atg cgt ttt gtt gct tca cct gat gag ctt cgc aaa tta gca aca 576Gly
Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr 180 185
190gaa gca tct cgt gaa gct gaa gcg gct ttc ggc gat ggc gcg gta tat
624Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr
195 200 205gtc gaa cgt gct gtg att aac cct cag cat att gaa gtg cag
atc ctt 672Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln
Ile Leu 210 215 220ggc gat cac act gga gaa gtt gta cac ctt tat gaa
cgt gac tgc tca 720Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu
Arg Asp Cys Ser225 230 235 240ctg cag cgt cgt cac caa aaa gtt gtc
gaa att gcg cca gca cag cat 768Leu Gln Arg Arg His Gln Lys Val Val
Glu Ile Ala Pro Ala Gln His 245 250 255ttg gat cca gaa ctg cgt gat
cgc att tgt gcg gat gca gta aag ttc 816Leu Asp Pro Glu Leu Arg Asp
Arg Ile Cys Ala Asp Ala Val Lys Phe 260 265 270tgc cgc tcc att ggt
tac cag ggc gcg gga acc gtg gaa ttc ttg gtc 864Cys Arg Ser Ile Gly
Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val 275 280 285gat gaa aag
ggc aac cac gtc ttc atc gaa atg aac cca cgt atc cag 912Asp Glu Lys
Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln 290 295 300gtt
gag cac acc gtg act gaa gaa gtc acc gag gtg gac ctg gtg aag 960Val
Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys305 310
315 320gcg cag atg cgc ttg gct gct ggt gca acc ttg aag gaa ttg ggt
ctg 1008Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly
Leu 325 330 335acc caa gat aag atc aag acc cac ggt gca gca ctg cag
tgc cgc atc 1056Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln
Cys Arg Ile 340 345 350acc acg gaa gat cca aac aac ggc ttc cgc cca
gat acc gga act atc 1104Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro
Asp Thr Gly Thr Ile 355 360 365acc gcg tac cgc tca cca ggc gga gct
ggc gtt cgt ctt gac ggt gca 1152Thr Ala Tyr Arg Ser Pro Gly Gly Ala
Gly Val Arg Leu Asp Gly Ala 370 375 380gct cag ctc ggt ggc gaa atc
acc gca cac ttt gac tcc atg ctg gtg 1200Ala Gln Leu Gly Gly Glu Ile
Thr Ala His Phe Asp Ser Met Leu Val385 390 395 400aaa atg acc tgc
cgt ggt tcc gac ttt gaa act gct gtt gct cgt gca 1248Lys Met Thr Cys
Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala 405 410 415cag cgc
gcg ttg gct gag ttc acc gtg tct ggt gtt gca acc aac att 1296Gln Arg
Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile 420 425
430ggt ttc ttg cgt gcg ttg ctg cgg gaa gag gac ttc act tcc aag cgc
1344Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445atc gcc acc gga ttc att gcc gat cac ccg cac ctc ctt cag
gct cca 1392Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln
Ala Pro 450 455 460cct gct gat gat gag cag gga cgc atc ctg gat tac
ttg gca gat gtc 1440Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr
Leu Ala Asp Val465 470 475 480acc gtg aac aag cct cat ggt gtg cgt
cca aag gat gtt gca gct cct 1488Thr Val Asn Lys Pro His Gly Val Arg
Pro Lys Asp Val Ala Ala Pro 485 490 495atc gat aag ctg cct aac atc
aag gat ctg cca ctg cca cgc ggt tcc 1536Ile Asp Lys Leu Pro Asn Ile
Lys Asp Leu Pro Leu Pro Arg Gly Ser 500 505 510cgt gac cgc ctg aag
cag ctt ggc cca gcc gcg ttt
gct cgt gat ctc 1584Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe
Ala Arg Asp Leu 515 520 525cgt gag cag gac gca ctg gca gtt act gat
acc acc ttc cgc gat gca 1632Arg Glu Gln Asp Ala Leu Ala Val Thr Asp
Thr Thr Phe Arg Asp Ala 530 535 540cac cag tct ttg ctt gcg acc cga
gtc cgc tca ttc gca ctg aag cct 1680His Gln Ser Leu Leu Ala Thr Arg
Val Arg Ser Phe Ala Leu Lys Pro545 550 555 560gcg gca gag gcc gtc
gca aag ctg act cct gag ctt ttg tcc gtg gag 1728Ala Ala Glu Ala Val
Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu 565 570 575gcc tgg ggc
ggc gcg acc tac gat gtg gcg atg cgt ttc ctc ttt gag 1776Ala Trp Gly
Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu 580 585 590gat
ccg tgg gac agg ctc gac gag ctg cgc gag gcg atg ccg aat gta 1824Asp
Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val 595 600
605aac att cag atg ctg ctt cgc ggc cgc aac acc gtg gga tac acc ccg
1872Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620tac cca gac tcc gtc tgc cgc gcg ttt gtt aag gaa gct gcc
agc tcc 1920Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala
Ser Ser625 630 635 640ggc gtg gac atc ttc cgc atc ttc gac gcg ctt
aac gac gtc tcc cag 1968Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu
Asn Asp Val Ser Gln 645 650 655atg cgt cca gca atc gac gca gtc ctg
gag acc aac acc gcg gta gcc 2016Met Arg Pro Ala Ile Asp Ala Val Leu
Glu Thr Asn Thr Ala Val Ala 660 665 670gag gtg gct atg gct tat tct
ggt gat ctc tct gat cca aat gaa aag 2064Glu Val Ala Met Ala Tyr Ser
Gly Asp Leu Ser Asp Pro Asn Glu Lys 675 680 685ctc tac acc ctg gat
tac tac cta aag atg gca gag gag atc gtc aag 2112Leu Tyr Thr Leu Asp
Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys 690 695 700tct ggc gct
cac atc ttg gcc att aag gat atg gct ggt ctg ctt cgc 2160Ser Gly Ala
His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg705 710 715
720cca gct gcg gta acc aag ctg gtc acc gca ctg cgc cgt gaa ttc gat
2208Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735ctg cca gtg cac gtg cac acc cac gac act gcg ggt ggc cag
ctg gca 2256Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln
Leu Ala 740 745 750acc tac ttt gct gca gct caa gct ggt gca gat gct
gtt gac ggt gct 2304Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala
Val Asp Gly Ala 755 760 765tcc gca cca ctg tct ggc acc acc tcc cag
cca tcc ctg tct gcc att 2352Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln
Pro Ser Leu Ser Ala Ile 770 775 780gtt gct gca ttc gcg cac acc cgt
cgc gat acc ggt ttg agc ctc gag 2400Val Ala Ala Phe Ala His Thr Arg
Arg Asp Thr Gly Leu Ser Leu Glu785 790 795 800gct gtt tct gac ctc
gag ccg tac tgg gaa gca gtg cgc gga ctg tac 2448Ala Val Ser Asp Leu
Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr 805 810 815ctg cca ttt
gag tct gga acc cca ggc cca acc ggt cgc gtc tac cgc 2496Leu Pro Phe
Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg 820 825 830cac
gaa atc cca ggc gga cag ttg tcc aac ctg cgt gca cag gcc acc 2544His
Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr 835 840
845gca ctg ggc ctt gcg gat cgt ttc gaa ctc atc gaa gac aac tac gca
2592Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860gcc gtt aat gag atg ctg gga cgc cca acc aag gtc acc cca
tcc tcc 2640Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro
Ser Ser865 870 875 880aag gtt gtt ggc gac ctc gca ctc cac ctc gtt
ggt gcg ggt gtg gat 2688Lys Val Val Gly Asp Leu Ala Leu His Leu Val
Gly Ala Gly Val Asp 885 890 895cca gca gac ttt gct gcc gat cca caa
aag tac gac atc cca gac tct 2736Pro Ala Asp Phe Ala Ala Asp Pro Gln
Lys Tyr Asp Ile Pro Asp Ser 900 905 910gtc atc gcg ttc ctg cgc ggc
gag ctt ggt aac cct cca ggt ggc tgg 2784Val Ile Ala Phe Leu Arg Gly
Glu Leu Gly Asn Pro Pro Gly Gly Trp 915 920 925cca gag cca ctg cgc
acc cgc gca ctg gaa ggc cgc tcc gaa ggc aag 2832Pro Glu Pro Leu Arg
Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys 930 935 940gca cct ctg
acg gaa gtt cct gag gaa gag cag gcg cac ctc gac gct 2880Ala Pro Leu
Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala945 950 955
960gat gat tcc aag gaa cgt cgc aat agc ctc aac cgc ctg ctg ttc ccg
2928Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975aag cca acc gaa gag ttc ctc gag cac cgt cgc cgc ttc ggc
aac acc 2976Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly
Asn Thr 980 985 990tct gcg ctg gat gat cgt gaa ttc ttc tac ggc ctg
gtc gaa ggc cgc 3024Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu
Val Glu Gly Arg 995 1000 1005gag act ttg atc cgc ctg cca gat gtg
cgc acc cca ctg ctt gtt cgc 3072Glu Thr Leu Ile Arg Leu Pro Asp Val
Arg Thr Pro Leu Leu Val Arg 1010 1015 1020ctg gat gcg atc tct gag
cca gac gat aag ggt atg cgc aat gtt gtg 3120Leu Asp Ala Ile Ser Glu
Pro Asp Asp Lys Gly Met Arg Asn Val Val1025 1030 1035 1040gcc aac
gtc aac ggc cag atc cgc cca atg cgt gtg cgt gac cgc tcc 3168Ala Asn
Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg Asp Arg Ser 1045 1050
1055gtt gag tct gtc acc gca acc gca gaa aag gca gat tcc tcc aac aag
3216Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp Ser Ser Asn Lys
1060 1065 1070ggc cat gtt gct gca cca ttc gct ggt gtt gtc acc gtg
act gtt gct 3264Gly His Val Ala Ala Pro Phe Ala Gly Val Val Thr Val
Thr Val Ala 1075 1080 1085gaa ggt gat gag gtc aag gct gga gat gca
gtc gca atc atc gag gct 3312Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
Val Ala Ile Ile Glu Ala 1090 1095 1100atg aag atg gaa gca aca atc
act gct tct gtt gac ggc aaa atc gat 3360Met Lys Met Glu Ala Thr Ile
Thr Ala Ser Val Asp Gly Lys Ile Asp1105 1110 1115 1120cgc gtt gtg
gtt cct gct gca acg aag gtg gaa ggt ggc gac ttg atc 3408Arg Val Val
Val Pro Ala Ala Thr Lys Val Glu Gly Gly Asp Leu Ile 1125 1130
1135gtc gtc gtt tcc taa 3423Val Val Val Ser
1140471140PRTCorynebacterium glutamicum 47Val Ser Thr His Thr Ser
Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu1 5 10 15Val Ala Asn Arg Gly
Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu 20 25 30Glu Thr Gly Ala
Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly 35 40 45Ser Phe His
Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu 50 55 60Gly Ser
Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala65 70 75
80Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile
Thr 100 105 110Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly
Asp Lys Ser 115 120 125Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu
Pro Val Leu Ala Glu 130 135 140Ser Thr Pro Ser Lys Asn Ile Asp Glu
Ile Val Lys Ser Ala Glu Gly145 150 155 160Gln Thr Tyr Pro Ile Phe
Val Lys Ala Val Ala Gly Gly Gly Gly Arg 165 170 175Gly Met Arg Phe
Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr 180 185 190Glu Ala
Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr 195 200
205Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp
Cys Ser225 230 235 240Leu Gln Arg Arg His Gln Lys Val Val Glu Ile
Ala Pro Ala Gln His 245 250 255Leu Asp Pro Glu Leu Arg Asp Arg Ile
Cys Ala Asp Ala Val Lys Phe 260 265 270Cys Arg Ser Ile Gly Tyr Gln
Gly Ala Gly Thr Val Glu Phe Leu Val 275 280 285Asp Glu Lys Gly Asn
His Val Phe Ile Glu Met Asn Pro Arg Ile Gln 290 295 300Val Glu His
Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys305 310 315
320Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys
Arg Ile 340 345 350Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp
Thr Gly Thr Ile 355 360 365Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly
Val Arg Leu Asp Gly Ala 370 375 380Ala Gln Leu Gly Gly Glu Ile Thr
Ala His Phe Asp Ser Met Leu Val385 390 395 400Lys Met Thr Cys Arg
Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala 405 410 415Gln Arg Ala
Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile 420 425 430Gly
Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg 435 440
445Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
450 455 460Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala
Asp Val465 470 475 480Thr Val Asn Lys Pro His Gly Val Arg Pro Lys
Asp Val Ala Ala Pro 485 490 495Ile Asp Lys Leu Pro Asn Ile Lys Asp
Leu Pro Leu Pro Arg Gly Ser 500 505 510Arg Asp Arg Leu Lys Gln Leu
Gly Pro Ala Ala Phe Ala Arg Asp Leu 515 520 525Arg Glu Gln Asp Ala
Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala 530 535 540His Gln Ser
Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro545 550 555
560Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu
Phe Glu 580 585 590Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala
Met Pro Asn Val 595 600 605Asn Ile Gln Met Leu Leu Arg Gly Arg Asn
Thr Val Gly Tyr Thr Pro 610 615 620Tyr Pro Asp Ser Val Cys Arg Ala
Phe Val Lys Glu Ala Ala Ser Ser625 630 635 640Gly Val Asp Ile Phe
Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln 645 650 655Met Arg Pro
Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala 660 665 670Glu
Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys 675 680
685Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu
Leu Arg705 710 715 720Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu
Arg Arg Glu Phe Asp 725 730 735Leu Pro Val His Val His Thr His Asp
Thr Ala Gly Gly Gln Leu Ala 740 745 750Thr Tyr Phe Ala Ala Ala Gln
Ala Gly Ala Asp Ala Val Asp Gly Ala 755 760 765Ser Ala Pro Leu Ser
Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile 770 775 780Val Ala Ala
Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu785 790 795
800Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val
Tyr Arg 820 825 830His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg
Ala Gln Ala Thr 835 840 845Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu
Ile Glu Asp Asn Tyr Ala 850 855 860Ala Val Asn Glu Met Leu Gly Arg
Pro Thr Lys Val Thr Pro Ser Ser865 870 875 880Lys Val Val Gly Asp
Leu Ala Leu His Leu Val Gly Ala Gly Val Asp 885 890 895Pro Ala Asp
Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser 900 905 910Val
Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp 915 920
925Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu
Asp Ala945 950 955 960Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn
Arg Leu Leu Phe Pro 965 970 975Lys Pro Thr Glu Glu Phe Leu Glu His
Arg Arg Arg Phe Gly Asn Thr 980 985 990Ser Ala Leu Asp Asp Arg Glu
Phe Phe Tyr Gly Leu Val Glu Gly Arg 995 1000 1005Glu Thr Leu Ile
Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val Arg 1010 1015 1020Leu
Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn Val Val1025
1030 1035 1040Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
Asp Arg Ser 1045 1050 1055Val Glu Ser Val Thr Ala Thr Ala Glu Lys
Ala Asp Ser Ser Asn Lys 1060 1065 1070Gly His Val Ala Ala Pro Phe
Ala Gly Val Val Thr Val Thr Val Ala 1075 1080 1085Glu Gly Asp Glu
Val Lys Ala Gly Asp Ala Val Ala Ile Ile Glu Ala 1090 1095 1100Met
Lys Met Glu Ala Thr Ile Thr Ala Ser Val Asp Gly Lys Ile Asp1105
1110 1115 1120Arg Val Val Val Pro Ala Ala Thr Lys Val Glu Gly Gly
Asp Leu Ile 1125 1130 1135Val Val Val Ser 1140
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